crystallization communications Acta Crystallographica Section F
Structural Biology Communications ISSN 2053-230X
Michael R. Oliver,a‡ Jennifer M. Crowther,a‡ Mary M. Leeman,b Sarah A. Kessans,a Rachel A. North,a Katherine A. Donovan,a Michael D. W. Griffin,c Hironori Suzuki,a Andre´ O. Hudson,b a Mu ¨ ge Kasanmascheff and Renwick C. J. Dobsona,c* a
Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand, bThe Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology (RIT), Rochester, New York, USA, and c Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria, Australia
‡ Joint first authors.
Correspondence e-mail: [email protected]
Received 18 December 2013 Accepted 7 April 2014
# 2014 International Union of Crystallography All rights reserved
Acta Cryst. (2014). F70, 663–668
The purification, crystallization and preliminary X-ray diffraction analysis of two isoforms of meso-diaminopimelate decarboxylase from Arabidopsis thaliana Diaminopimelate decarboxylase catalyses the last step in the diaminopimelatebiosynthetic pathway leading to S-lysine: the decarboxylation of mesodiaminopimelate to form S-lysine. Lysine biosynthesis occurs only in microorganisms and plants, and lysine is essential for the growth and development of animals. Thus, the diaminopimelate pathway represents an attractive target for antimicrobial and herbicide treatments and has received considerable attention from both a mechanistic and a structural viewpoint. Diaminopimelate decarboxylase has only been characterized in prokaryotic species. This communication describes the first structural studies of two diaminopimelate decarboxylase isoforms from a plant. The Arabidopsis thaliana diaminopimelate decarboxylase cDNAs At3g14390 (encoding DapDc1) and At5g11880 (encoding DapDc2) were cloned from genomic DNA and the recombinant proteins were expressed and purified from Escherichia coli Rosetta (DE3) cells. The crystals of DapDc1 and DapDc2 diffracted to beyond 2.00 ˚ resolution, respectively. Understanding the structural biology of and 2.27 A diaminopimelate decarboxylase from a eukaryotic species will provide insights for the development of future herbicide treatments, in particular. 1. Introduction S-Lysine is an amino acid that is required by living organisms for protein synthesis (Azevedo et al., 1997). In addition, many bacteria use lysine, or its precursor diaminopimelate, as a component of their peptidoglycan cell wall, which is necessary for maintaining the structural integrity of the cell (Dogovski et al., 2012; McGroty et al., 2013). Animals do not possess the enzymes for synthesizing lysine de novo and must obtain it through their diet, while plants and bacteria rely on their own production of the amino acid for growth (Jander & Joshi, 2009). Therefore, the enzymes involved in this pathway are attractive targets for antibiotics and herbicides (Boughton, Dobson et al., 2008; Boughton, Griffin et al., 2008; Mitsakos et al., 2008). Two pathways have evolved to synthesize lysine: the diaminopimelate pathway and the -aminoadipic acid pathway (Velasco et al., 2002). Although plants and bacteria utilize variants of the diaminopimelate pathway (McCoy et al., 2006; Hudson et al., 2005), the final step is shared in each variant: the decarboxylation of meso-diaminopimelate to yield lysine (see Fig. 1). This last step is catalysed by the enzyme diaminopimelate decarboxylase (DapDc). DapDc is highly specific for the meso isomer of diaminopimelate over the R,R and S,S isomers (Kelland et al., 1986; Cox et al., 2000). During decarboxylation a bond is broken between the C atom and the carboxyl C atom exclusively at the d stereocentre of mesodiaminopimelate, specifically yielding S-lysine (Kelland et al., 1986; Ray et al., 2002). DapDc is the only known amino-acid decarboxylase that stereospecifically acts on a chiral C atom in the R configuration (Gokulan et al., 2003), with all known pyridoxal phosphate (PLP)dependent decarboxylases and human amino-acid decarboxylases acting on S substrates (Ray et al., 2002). Therefore, cross-inhibition of similar enzymes by diaminopimelate decarboxylase inhibitors is less likely, reducing the risk to humans. Two genes from the genome of Arabidopsis thaliana, At3g14390 and At5g11880, have been identified as encoding DapDc1 and DapDc2, respectively (Hudson et al., 2005). While sharing 93% sequence identity with each other, these plant homologues of DapDc doi:10.1107/S2053230X14007699
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crystallization communications show a low degree of sequence identity (37–27%) compared with the archaeal and bacterial homologues for which structures have been solved and deposited in the Protein Data Bank (PDB entries 1knw, 3c5q, 2qgh, 2yxx, 3n2b, 1lo0, 1hkv, 1tuf, 2o0t, 1twi, 2j66 and 2p3e; Fig. 2). The deposited structures, however, reveal a high level of structural similarity between homologues but some differences in quaternary structure. To date, no crystal structures of plant DapDc homologues have been reported. Here, we present the cloning, expression, purification and crystallization of the A. thaliana DapDc1 and DapDc2 isoforms.
2. Materials and methods 2.1. Cloning, expression and purification
Total RNA was isolated from seven-day-old Col-7 A. thaliana seedlings using TRIzol reagent (Life Technologies; Prabhu & Hudson, 2010). cDNA synthesis was performed using a reverse transcription system kit (Promega) with 1.0 mg of total RNA following the manufacturer’s protocol. The cDNAs of At3g14390 and At5g11880, which encode DapDc1 and DapDc2, respectively, were amplified by PCR using 12 pmol forward and reverse primers, 1 mM MgSO4, 0.5 mM of each of the four deoxynucleotide triphosphates, 2 ml cDNA product and one unit of Platinum Pfx DNA polymerase (Invitrogen) using the following PCR conditions: one cycle at 94 C
for 2 min followed by 30 cycles of 94 C for 15 s, 60 C for 30 s and 72 C for 2 min. The forward and reverse primers used to amplify the At3g14390 cDNA were 50 -CCCCCGGATCCATGGCCGCCGTCGTTTCTCAAAACTCGTCC-30 and 50 -CCCCCGTCGACTCATAGACCTTCAAAGAAACGCAAATGGTC-30 . The forward and reverse primers used to amplify the At5g11880 cDNA were 50 -CCCCCGGATCCATGGCCGCCGTTTCCCAAAACTCCTACCAAA-30 and 50 -CCCCCGTCGACTCATAGTCCTTCAAAGAAACGTAAATGGTC-30 . The underlined nucleotides represent the restriction-enzyme sites used in the cloning of the cDNAs, while the bold and italicized nucleotides represent initiation and termination codons. It should be noted that the PCR was designed to exclude the first 144 and 147 nucleotides from the At3g14390 and At5g11880 cDNAs, respectively, as these nucleotides encode a predicted transit peptide that allows localization to the chloroplast. The PCR fragment was sequenced to confirm the fidelity of the PCR reaction. For cloning, the PCR amplicons were digested with BamHI and SalI and ligated into the plasmid pET-30a (Novagen) to yield the plasmids pET-30a:: At3g14390 and pET-30a::At5g11880. The recombinant proteins derived from these plasmids carry a hexahistidine and S-tag epitope derived from the pET-30a plasmid at the amino-terminus. The resulting expression vectors, pET-30a::At3g14390 and pET30a::At5g11880, were transformed into Escherichia coli Rosetta (DE3) cells. The transformed cells were grown in 1 l LB medium (with 50 mg ml1 kanamycin and 34 mg ml1 chloramphenicol) for
Figure 1 Lysine-biosynthesis pathway (adapted from McCoy et al., 2006).
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crystallization communications approximately 4 h at 310 K until the OD600 reached 0.5. Expression at 299 K overnight was induced by the addition of 0.5 mM isopropyl -d-1-thiogalactopyranoside. The cell pellet was resuspended in buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole pH 8) and lysed by sonication using a cell disruptor (Vibra-Cell VC 750; Sonics). The lysate was clarified by centrifugation (12 000g, 277 K, 10 min) and loaded onto a GE Healthcare His-Trap FF Crude 5 ml column (GE Healthcare) preequilibrated with buffer A. The bound His-tagged protein was eluted with an increasing imidazole concentration as buffer A was replaced with buffer B (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole pH 8). Fractions containing protein were pooled and concentrated to 3 ml using a 30 kDa molecular-weight cutoff spin concentrator (Vivaspin centrifugal concentrator, Sartorius). The concentrated
solution was further purified on a HiLoad 10/30 Superdex 200 column (GE Healthcare) pre-equilibrated with buffer C (20 mM Tris–HCl pH 8, 100 mM NaCl). All purification steps were carried out at 277 K. Peak fractions containing DapDc1 and DapDc2 were identified by SDS–PAGE using BOLT Bis-Tris Plus Gels (Life Technologies, USA) and concentrated to 10 mg ml1 using a 30 kDa molecular-weight cutoff spin concentrator. Protein concentration was determined by the Bradford assay (Bradford, 1976). 2.2. Sequence analysis of two isoforms of plant DapDc
A multiple protein sequence alignment was performed between the plant DapDc isoforms DapDc1 (Q949X7) and DapDc2 (Q94A94), one archaeal species (Methanococcus jannaschii strain
Figure 2 Sequence alignment of diaminopimelate decarboxylase from A. thaliana (DapDc1 and DapDc2), one archaeal species (Methanococcus jannaschii) and eight bacterial species (Mycobacterium tuberculosis, Helicobacter pylori, Vibrio cholerae, Thermotoga maritima, Aquifex aeolicus, Bacillus circulans, Escherichia coli and Brucella melitensis). Conserved residues are highlighted in pink.
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crystallization communications ATCC 43067; Q58497) and eight eubacterial species [Mycobacterium tuberculosis (P0A5M4), Helicobacter pylori (B4XMC6), Vibrio cholerae serotype O1 (Q9KVL7), Thermotoga maritima strain ATCC 43589 (Q9X1K5), Aquifex aeolicus strain VF5 (O67262), Bacillus circulans (Q2L4H3), E. coli strain K12 (P00861) and Brucella melitensis strain M28 (F2HSW8)] (extracted from http://www.uniprot.org; protein accession numbers are given in parentheses). The alignment was performed using the program MUSCLE (Edgar, 2004), with manual inspection and editing. 2.3. Crystallization 2.3.1. Crystallization of DapDc1. Crystallization studies were initially conducted using two different concentrations of DapDc1, 10 and 5 mg ml1, in buffer C containing 0.2 mM PLP and 0.4 mM lysine. Initial sitting-drop vapour-diffusion crystallization trials were performed using a Mosquito crystallization robot (TTP Labtech). The commercial crystallization screens PACT, JCSG+ and Clear Strategy Screens 1 and 2 (Molecular Dimensions Ltd) were used to identify crystallization conditions at 277 and 293 K. Crystallization drops of 800 nl (protein solution:reservoir solution ratio of 1:1) were set up in 96-well plates containing 50 ml reservoir solution. Initial needle-like shaped crystals were obtained after 3–6 d from several of the PACT (E2, D10, H2 and H10) and JCSG+ (E2 and G11) screen conditions. Optimization of the initial hits was carried out by altering the protein concentration (5, 8 and 10 mg ml1), the pH (5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) and the PEG concentration [14, 16, 18, 20 and 22%(w/v)] of the following PACT conditions: D10 [0.2 M magnesium chloride, 0.1 M Tris pH 8, 20%(w/v) PEG 6000] and H2 [0.2 M sodium bromide, 0.1 M bis-tris propane pH 8.5, 20%(w/v) PEG 3350]. The best diffracting needle-shaped crystal of DapDc1 was obtained from the following sitting-drop condition: 8 mg ml1 protein in buffer C containing 0.2 mM PLP and 0.4 mM lysine was mixed in a 1:1 ratio with a reservoir solution consisting of 0.2 M magnesium chloride, 0.1 M Tris pH 7.5, 20%(w/v) PEG 6000. The drop volume was 800 nl and the reservoir volume was 100 ml. 2.3.2. Crystallization of DapDc2. Crystallization studies were initially conducted using two different conditions at 10 mg ml1 DapDc2: (i) in buffer C and (ii) in buffer C containing 0.2 mM PLP and 5 mM lysine. Initial protein crystallization trials were performed at the CSIRO node of the Bio21 Collaborative Crystallization Centre (C3; http://www.csiro.au/c3/) using the PACT and JSCG+ screens (Newman et al., 2005, 2008) at 281 and 293 K as described previously
(Dobson et al., 2008). Crystallization screens were performed using the sitting-drop vapour-diffusion method with droplets consisting of 150 nl protein solution and 150 nl reservoir solution. Initial plateshaped crystals were obtained after 2–5 d from the PACT (B7, C9, C10, F1, F2, F3, F5 and F7) and JCSG+ (A5 and G10) screen conditions. Optimization of the initial hits was carried out by altering the protein concentration (5, 8 and 10 mg ml1), the salt concentration in the reservoir solution (0.22, 0.24, 0.26, 0.28 and 0.30 M), the pH (5.0, 5.5, 6.0, 6.5, 7.0 and 7.5) and the PEG concentration [14, 16, 18, 20, 22 and 25%(w/v)] of PACT condition F7, which consists of 0.2 M sodium acetate, 0.1 M bis-tris propane pH 6.5, 20%(w/v) PEG 3350. The best diffracting DapDc2 crystal was obtained from the following condition: 8 mg ml1 protein in buffer C containing 0.2 mM PLP and 5 mM lysine mixed in a 1:1 ratio with a reservoir solution consisting of 0.28 M sodium acetate, 0. M bis-tris propane pH 7.2, 25%(w/v) PEG 3350. The drop volume was 800 nl and the reservoir volume was 100 ml. 2.4. Data collection and processing
For data collection, both DapDc1 and DapDc2 crystals (Fig. 3) were briefly soaked in cryoprotectant solution which consisted of 85%(v/v) reservoir solution and 15%(v/v) 1:1 ethylene glycol:glycerol solution. The crystals were harvested with a litho-loop and flashcooled in liquid nitrogen. Diffraction data were collected at 110 K on the MX2 beamline at the Australian Synchrotron, Victoria, Australia ˚ . The detector was positioned 280 and at a wavelength of 0.95369 A 300 mm from the DapDc1 and DapDc2 crystals, respectively. The data for the DapDc1 and DapDC2 crystals were collected in 0.5 steps over one 180 pass with exposure times of 1 and 0.5 s, respectively. Diffraction images were indexed and integrated using XDS (Kabsch, 2010). Scaling and data reduction were then performed using SCALA (Evans, 2006) and AIMLESS from the CCP4 suite (Winn et al., 2011). All relevant data-collection and processing parameters are given in Table 1.
3. Results and discussion The plant DapDc isoforms DapDc1 (coded by At3g14390) and DapDc2 (coded by At5g11880) were purified to homogeneity using Ni2+-affinity chromatography and size-exclusion chromatography. From a 1 l bacterial cell culture, 40 mg of protein was obtained.
Figure 3 Crystals of DapDc1 (left) and DapDc2 (right). The scale bar indicates 0.1 mm.
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crystallization communications Analysis of the proteins by SDS–PAGE and Coomassie Brilliant Blue staining showed a purity of at least 95% (Fig. 4). A multiple sequence alignment was carried out between the two homologues of diaminopimelate decarboxylase from A. thaliana (DapDc1 and DapDc2), eight additional bacterial species and a single archaeal species. The sequence alignment, shown in Fig. 2, illustrates the residues that are conserved between species, which are likely to Table 1 X-ray data-collection statistics for the two isoforms of plant diaminopimelate decarboxylase DapDc1 and DapDc2. Values in parentheses are for the highest resolution shell. The Matthews coefficients and solvent contents are based on two monomers.
˚) Wavelength (A No. of images Oscillation ( ) Space group ˚ , ) Unit-cell parameters (A ˚) Resolution (A Observed reflections Unique reflections Completeness (%) Rmerge† Rr.i.m.‡ Rp.i.m.§ Mean I/(I) Multiplicity ˚ 2) Wilson B value (A Molecules per asymmetric unit ˚ 3 Da1) VM (A Estimated solvent content (%)
DapDc1
DapDc2
0.95369 360 0.5 P21212 a = 80.9, b = 121.5, c = 88.6, = = = 90 48.62–2.00 (2.11–2.00) 440743 (64183) 59879 (8647) 100.00 (100.00) 0.129 (0.673) 0.138 (0.724) 0.051 (0.265) 11.3 (2.9) 7.4 (7.4) 26.4 2 2.22 45
0.95369 360 0.5 P21 a = 51.3, b = 107.9, c = 80.8, = = 90, = 98.6 46.02–2.27 (2.35–2.27) 151241 (13677) 39937 (3640) 99.8 (98.1) 0.155 (0.650) 0.213 (0.889) 0.145 (0.604) 6.9 (2.0) 3.8 (3.8) 26.1 2 2.34 48
P P P P †PRmerge = ‡ Rr.i.m. = hkl i jIi ðhklÞ hIðhklÞij=P hkl P i Ii ðhklÞ. 1=2 P 1g = i jIi ðhklÞ hIðhklÞij= Phkl fNðhklÞ=½NðhklÞ1=2 P P hkl i Ii ðhklÞ. § Rp.i.m. P hkl f1=½NðhklÞ 1g i jIi ðhklÞ hIðhklÞij= hkl i Ii ðhklÞ, where Ii(hkl) is the ith intensity measurement of reflection hkl, hI(hkl)i is its average and N(hkl) is the redundancy of a given reflection.
Figure 4 Purification of the recombinant plant diaminopimelate decarboxylase isoforms DapDc1 and DapDc2. (a) SDS–PAGE gel showing purification of the DapDc1 isoform. Lane 1, molecular-weight markers (labelled in kDa); lane 2, crude cell lysate; lane 3, pooled fractions after immobilized metal-affinity chromatography; lane 4, post size-exclusion chromatography. (b) SDS–PAGE gel showing purification of the DapDc2 isoform. Lane 1, crude cell lysate; lane 2, pooled fractions after immobilized metal-affinity chromatography; lane 3, post sizeexclusion chromatography; lane 4, molecular-weight markers (labelled in kDa).
Acta Cryst. (2014). F70, 663–668
be important for protein function. Two consensus motifs for PLP binding have previously been identified in diaminopimelate decarboxylase from A. thaliana (Kim, 2006). The first motif includes a conserved lysine residue, Lys67 (amino-acid numbering corresponds to that DapDc1), which forms a Schiff base with the aldehyde moiety of PLP (Gokulan et al., 2003). The second motif features three consecutive glycine residues, Gly244–Gly246, which are hypothesized to interact with the phosphate moiety of the bound cofactor (Momany et al., 1995). Substrate binding is mediated via a salt bridge between the substrate and arginine residue Arg321 (Ray et al., 2002). A second arginine, Arg285, is also within bonding distance. Both of these arginines are conserved across all of the aligned species. Furthermore, two residues, Cys353 and Glu282, which are hypothesized to a play a catalytic role are both conserved (Jackson et al., 2000). The conservation of these residues within the active site suggest that the plant homologues of diaminopimelate decarboxylase utilize the same active-site architecture as prokaryotic homologues of the enzyme to achieve substrate and reaction specificity. The PACT screen conditions D10 and H2 produced DapDc1 crystals and condition F7 produced DapDc2 crystals. In-house optimization resulted in larger sized crystals (see xx2.3.1 and 2.3.2 for details). The best diffracting crystals of DapDc1 and DapDC2 were needle-shaped crystals of lengths 0.1 and 0.05 mm, respectively, that ˚ grew within 4 d at 293 K (Fig. 3). Complete data sets to 2.00 A ˚ resolution for DapDc1 and to 2.27 A resolution for DapDc2 were collected from these crystals. Data-collection and processing statistics are summarized in Table 1. Preliminary diffraction data analysis using XDS indicated that the DapDc1 and DapDc2 crystals belonged to space groups P21212 and P21, respectively. Initial phases were estimated by molecular replacement using MOLREP and Phaser from the CCP4 suite. The monomer of A. aeolicus DapDc, which shares the highest sequence identity with both isoforms of the plant DapDc (37%), was used as the search model (PDB entry 2p3e; Southeast Collaboratory for Structural Genomics/RIKEN Structural Genomics/Proteomics Initiative, unpublished work). The initial round of refinement after molecular replacement resulted in a decrease in the R factor from 0.5448 to 0.4739 and in Rfree from 0.5499 to 0.5215 for DapDc1 and a decrease in the R factor from 0.4889 to 0.4050 and in Rfree from 0.4804 to 0.4512 for DapDc2. In each case the majority of the model fits the electron density well, which confirms solution of the crystal structure by molecular replacement, while a few regions, such as the terminal regions, do not fit so well. This results in relatively high Rfree values, but we are continuing to refine these models. The analysis suggested two DapDc1 molecules per asymmetric unit with a Matthews ˚ 3 Da1 and a solvent content coefficient (Matthews, 1968) of 2.22 A of 45% and two DapDc2 molecules per asymmetric unit with a ˚ 3 Da1 and a solvent Matthews coefficient (Matthews, 1968) of 2.34 A content of 48%. These findings suggest that DapDc1 and DapDc2 may exist as dimers. This is in agreement with other studies that suggest that the diaminopimelate decarboxylases from M. jannaschii (Ray et al., 2002), H. pylori (Hu et al., 2008) and M. tuberculosis (Gokulan et al., 2003) also form dimers. The structures of the two isomers of plant DapDc will be published elsewhere. In summary, we present the purification, crystallization and preliminary X-ray diffraction analysis of two DapDc isomers from A. thaliana, the first eukaryotic DapDc homologue studied thus far. The structural information gathered from the characterization of plant DapDc may be used to identify specific differences between plant and bacterial homologues of this lysine-biosynthetic enzyme. This information could be useful in the design of herbicide inhibitors that selectively target plant DapDc. Oliver et al.
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crystallization communications We acknowledge the support and assistance of the friendly staff at the CSIRO Collaborative Crystallization Centre at CSIRO Material Science and Engineering, Parkville, Melbourne and the MX beamline scientists at the Australian Synchrotron, Victoria, Australia. Parts of this research were undertaken at the MX2 beamline of the Australian Synchrotron, Victoria, Australia. Travel to the Australian Synchrotron was supported by the New Zealand Synchrotron Group. HS acknowledges the 2012 Researcher Exchange Program between JSPS and RSNZ. RCJD acknowledges (i) the New Zealand Royal Society Marsden Fund for funding support, in part (contract UOC1013), and (ii) the US Army Research Laboratory and US Army Research Office under contract/grant No. W911NF-11-1-0481 for support, in part. AOH acknowledges a National Science Foundation (NSF) award (MCB-#1120541) and a Rochester Institute of Technology (RIT) College of Science 2012 Dean’s Research Initiation Grant. MML was supported by the NSF award to AOH. MDWG is the recipient of the C. R. Roper Fellowship. We especially thank Jackie Healy for her technical support.
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Structural Biology Communications ISSN 2053-230X
Michael R. Oliver,a‡ Jennifer M. Crowther,a‡ Mary M. Leeman,b Sarah A. Kessans,a Rachel A. North,a Katherine A. Donovan,a Michael D. W. Griffin,c Hironori Suzuki,a Andre´ O. Hudson,b a Mu ¨ ge Kasanmascheff and Renwick C. J. Dobsona,c* a
Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand, bThe Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology (RIT), Rochester, New York, USA, and c Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria, Australia
‡ Joint first authors.
Correspondence e-mail: [email protected]
Received 18 December 2013 Accepted 7 April 2014
# 2014 International Union of Crystallography All rights reserved
Acta Cryst. (2014). F70, 663–668
The purification, crystallization and preliminary X-ray diffraction analysis of two isoforms of meso-diaminopimelate decarboxylase from Arabidopsis thaliana Diaminopimelate decarboxylase catalyses the last step in the diaminopimelatebiosynthetic pathway leading to S-lysine: the decarboxylation of mesodiaminopimelate to form S-lysine. Lysine biosynthesis occurs only in microorganisms and plants, and lysine is essential for the growth and development of animals. Thus, the diaminopimelate pathway represents an attractive target for antimicrobial and herbicide treatments and has received considerable attention from both a mechanistic and a structural viewpoint. Diaminopimelate decarboxylase has only been characterized in prokaryotic species. This communication describes the first structural studies of two diaminopimelate decarboxylase isoforms from a plant. The Arabidopsis thaliana diaminopimelate decarboxylase cDNAs At3g14390 (encoding DapDc1) and At5g11880 (encoding DapDc2) were cloned from genomic DNA and the recombinant proteins were expressed and purified from Escherichia coli Rosetta (DE3) cells. The crystals of DapDc1 and DapDc2 diffracted to beyond 2.00 ˚ resolution, respectively. Understanding the structural biology of and 2.27 A diaminopimelate decarboxylase from a eukaryotic species will provide insights for the development of future herbicide treatments, in particular. 1. Introduction S-Lysine is an amino acid that is required by living organisms for protein synthesis (Azevedo et al., 1997). In addition, many bacteria use lysine, or its precursor diaminopimelate, as a component of their peptidoglycan cell wall, which is necessary for maintaining the structural integrity of the cell (Dogovski et al., 2012; McGroty et al., 2013). Animals do not possess the enzymes for synthesizing lysine de novo and must obtain it through their diet, while plants and bacteria rely on their own production of the amino acid for growth (Jander & Joshi, 2009). Therefore, the enzymes involved in this pathway are attractive targets for antibiotics and herbicides (Boughton, Dobson et al., 2008; Boughton, Griffin et al., 2008; Mitsakos et al., 2008). Two pathways have evolved to synthesize lysine: the diaminopimelate pathway and the -aminoadipic acid pathway (Velasco et al., 2002). Although plants and bacteria utilize variants of the diaminopimelate pathway (McCoy et al., 2006; Hudson et al., 2005), the final step is shared in each variant: the decarboxylation of meso-diaminopimelate to yield lysine (see Fig. 1). This last step is catalysed by the enzyme diaminopimelate decarboxylase (DapDc). DapDc is highly specific for the meso isomer of diaminopimelate over the R,R and S,S isomers (Kelland et al., 1986; Cox et al., 2000). During decarboxylation a bond is broken between the C atom and the carboxyl C atom exclusively at the d stereocentre of mesodiaminopimelate, specifically yielding S-lysine (Kelland et al., 1986; Ray et al., 2002). DapDc is the only known amino-acid decarboxylase that stereospecifically acts on a chiral C atom in the R configuration (Gokulan et al., 2003), with all known pyridoxal phosphate (PLP)dependent decarboxylases and human amino-acid decarboxylases acting on S substrates (Ray et al., 2002). Therefore, cross-inhibition of similar enzymes by diaminopimelate decarboxylase inhibitors is less likely, reducing the risk to humans. Two genes from the genome of Arabidopsis thaliana, At3g14390 and At5g11880, have been identified as encoding DapDc1 and DapDc2, respectively (Hudson et al., 2005). While sharing 93% sequence identity with each other, these plant homologues of DapDc doi:10.1107/S2053230X14007699
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crystallization communications show a low degree of sequence identity (37–27%) compared with the archaeal and bacterial homologues for which structures have been solved and deposited in the Protein Data Bank (PDB entries 1knw, 3c5q, 2qgh, 2yxx, 3n2b, 1lo0, 1hkv, 1tuf, 2o0t, 1twi, 2j66 and 2p3e; Fig. 2). The deposited structures, however, reveal a high level of structural similarity between homologues but some differences in quaternary structure. To date, no crystal structures of plant DapDc homologues have been reported. Here, we present the cloning, expression, purification and crystallization of the A. thaliana DapDc1 and DapDc2 isoforms.
2. Materials and methods 2.1. Cloning, expression and purification
Total RNA was isolated from seven-day-old Col-7 A. thaliana seedlings using TRIzol reagent (Life Technologies; Prabhu & Hudson, 2010). cDNA synthesis was performed using a reverse transcription system kit (Promega) with 1.0 mg of total RNA following the manufacturer’s protocol. The cDNAs of At3g14390 and At5g11880, which encode DapDc1 and DapDc2, respectively, were amplified by PCR using 12 pmol forward and reverse primers, 1 mM MgSO4, 0.5 mM of each of the four deoxynucleotide triphosphates, 2 ml cDNA product and one unit of Platinum Pfx DNA polymerase (Invitrogen) using the following PCR conditions: one cycle at 94 C
for 2 min followed by 30 cycles of 94 C for 15 s, 60 C for 30 s and 72 C for 2 min. The forward and reverse primers used to amplify the At3g14390 cDNA were 50 -CCCCCGGATCCATGGCCGCCGTCGTTTCTCAAAACTCGTCC-30 and 50 -CCCCCGTCGACTCATAGACCTTCAAAGAAACGCAAATGGTC-30 . The forward and reverse primers used to amplify the At5g11880 cDNA were 50 -CCCCCGGATCCATGGCCGCCGTTTCCCAAAACTCCTACCAAA-30 and 50 -CCCCCGTCGACTCATAGTCCTTCAAAGAAACGTAAATGGTC-30 . The underlined nucleotides represent the restriction-enzyme sites used in the cloning of the cDNAs, while the bold and italicized nucleotides represent initiation and termination codons. It should be noted that the PCR was designed to exclude the first 144 and 147 nucleotides from the At3g14390 and At5g11880 cDNAs, respectively, as these nucleotides encode a predicted transit peptide that allows localization to the chloroplast. The PCR fragment was sequenced to confirm the fidelity of the PCR reaction. For cloning, the PCR amplicons were digested with BamHI and SalI and ligated into the plasmid pET-30a (Novagen) to yield the plasmids pET-30a:: At3g14390 and pET-30a::At5g11880. The recombinant proteins derived from these plasmids carry a hexahistidine and S-tag epitope derived from the pET-30a plasmid at the amino-terminus. The resulting expression vectors, pET-30a::At3g14390 and pET30a::At5g11880, were transformed into Escherichia coli Rosetta (DE3) cells. The transformed cells were grown in 1 l LB medium (with 50 mg ml1 kanamycin and 34 mg ml1 chloramphenicol) for
Figure 1 Lysine-biosynthesis pathway (adapted from McCoy et al., 2006).
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crystallization communications approximately 4 h at 310 K until the OD600 reached 0.5. Expression at 299 K overnight was induced by the addition of 0.5 mM isopropyl -d-1-thiogalactopyranoside. The cell pellet was resuspended in buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole pH 8) and lysed by sonication using a cell disruptor (Vibra-Cell VC 750; Sonics). The lysate was clarified by centrifugation (12 000g, 277 K, 10 min) and loaded onto a GE Healthcare His-Trap FF Crude 5 ml column (GE Healthcare) preequilibrated with buffer A. The bound His-tagged protein was eluted with an increasing imidazole concentration as buffer A was replaced with buffer B (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole pH 8). Fractions containing protein were pooled and concentrated to 3 ml using a 30 kDa molecular-weight cutoff spin concentrator (Vivaspin centrifugal concentrator, Sartorius). The concentrated
solution was further purified on a HiLoad 10/30 Superdex 200 column (GE Healthcare) pre-equilibrated with buffer C (20 mM Tris–HCl pH 8, 100 mM NaCl). All purification steps were carried out at 277 K. Peak fractions containing DapDc1 and DapDc2 were identified by SDS–PAGE using BOLT Bis-Tris Plus Gels (Life Technologies, USA) and concentrated to 10 mg ml1 using a 30 kDa molecular-weight cutoff spin concentrator. Protein concentration was determined by the Bradford assay (Bradford, 1976). 2.2. Sequence analysis of two isoforms of plant DapDc
A multiple protein sequence alignment was performed between the plant DapDc isoforms DapDc1 (Q949X7) and DapDc2 (Q94A94), one archaeal species (Methanococcus jannaschii strain
Figure 2 Sequence alignment of diaminopimelate decarboxylase from A. thaliana (DapDc1 and DapDc2), one archaeal species (Methanococcus jannaschii) and eight bacterial species (Mycobacterium tuberculosis, Helicobacter pylori, Vibrio cholerae, Thermotoga maritima, Aquifex aeolicus, Bacillus circulans, Escherichia coli and Brucella melitensis). Conserved residues are highlighted in pink.
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crystallization communications ATCC 43067; Q58497) and eight eubacterial species [Mycobacterium tuberculosis (P0A5M4), Helicobacter pylori (B4XMC6), Vibrio cholerae serotype O1 (Q9KVL7), Thermotoga maritima strain ATCC 43589 (Q9X1K5), Aquifex aeolicus strain VF5 (O67262), Bacillus circulans (Q2L4H3), E. coli strain K12 (P00861) and Brucella melitensis strain M28 (F2HSW8)] (extracted from http://www.uniprot.org; protein accession numbers are given in parentheses). The alignment was performed using the program MUSCLE (Edgar, 2004), with manual inspection and editing. 2.3. Crystallization 2.3.1. Crystallization of DapDc1. Crystallization studies were initially conducted using two different concentrations of DapDc1, 10 and 5 mg ml1, in buffer C containing 0.2 mM PLP and 0.4 mM lysine. Initial sitting-drop vapour-diffusion crystallization trials were performed using a Mosquito crystallization robot (TTP Labtech). The commercial crystallization screens PACT, JCSG+ and Clear Strategy Screens 1 and 2 (Molecular Dimensions Ltd) were used to identify crystallization conditions at 277 and 293 K. Crystallization drops of 800 nl (protein solution:reservoir solution ratio of 1:1) were set up in 96-well plates containing 50 ml reservoir solution. Initial needle-like shaped crystals were obtained after 3–6 d from several of the PACT (E2, D10, H2 and H10) and JCSG+ (E2 and G11) screen conditions. Optimization of the initial hits was carried out by altering the protein concentration (5, 8 and 10 mg ml1), the pH (5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) and the PEG concentration [14, 16, 18, 20 and 22%(w/v)] of the following PACT conditions: D10 [0.2 M magnesium chloride, 0.1 M Tris pH 8, 20%(w/v) PEG 6000] and H2 [0.2 M sodium bromide, 0.1 M bis-tris propane pH 8.5, 20%(w/v) PEG 3350]. The best diffracting needle-shaped crystal of DapDc1 was obtained from the following sitting-drop condition: 8 mg ml1 protein in buffer C containing 0.2 mM PLP and 0.4 mM lysine was mixed in a 1:1 ratio with a reservoir solution consisting of 0.2 M magnesium chloride, 0.1 M Tris pH 7.5, 20%(w/v) PEG 6000. The drop volume was 800 nl and the reservoir volume was 100 ml. 2.3.2. Crystallization of DapDc2. Crystallization studies were initially conducted using two different conditions at 10 mg ml1 DapDc2: (i) in buffer C and (ii) in buffer C containing 0.2 mM PLP and 5 mM lysine. Initial protein crystallization trials were performed at the CSIRO node of the Bio21 Collaborative Crystallization Centre (C3; http://www.csiro.au/c3/) using the PACT and JSCG+ screens (Newman et al., 2005, 2008) at 281 and 293 K as described previously
(Dobson et al., 2008). Crystallization screens were performed using the sitting-drop vapour-diffusion method with droplets consisting of 150 nl protein solution and 150 nl reservoir solution. Initial plateshaped crystals were obtained after 2–5 d from the PACT (B7, C9, C10, F1, F2, F3, F5 and F7) and JCSG+ (A5 and G10) screen conditions. Optimization of the initial hits was carried out by altering the protein concentration (5, 8 and 10 mg ml1), the salt concentration in the reservoir solution (0.22, 0.24, 0.26, 0.28 and 0.30 M), the pH (5.0, 5.5, 6.0, 6.5, 7.0 and 7.5) and the PEG concentration [14, 16, 18, 20, 22 and 25%(w/v)] of PACT condition F7, which consists of 0.2 M sodium acetate, 0.1 M bis-tris propane pH 6.5, 20%(w/v) PEG 3350. The best diffracting DapDc2 crystal was obtained from the following condition: 8 mg ml1 protein in buffer C containing 0.2 mM PLP and 5 mM lysine mixed in a 1:1 ratio with a reservoir solution consisting of 0.28 M sodium acetate, 0. M bis-tris propane pH 7.2, 25%(w/v) PEG 3350. The drop volume was 800 nl and the reservoir volume was 100 ml. 2.4. Data collection and processing
For data collection, both DapDc1 and DapDc2 crystals (Fig. 3) were briefly soaked in cryoprotectant solution which consisted of 85%(v/v) reservoir solution and 15%(v/v) 1:1 ethylene glycol:glycerol solution. The crystals were harvested with a litho-loop and flashcooled in liquid nitrogen. Diffraction data were collected at 110 K on the MX2 beamline at the Australian Synchrotron, Victoria, Australia ˚ . The detector was positioned 280 and at a wavelength of 0.95369 A 300 mm from the DapDc1 and DapDc2 crystals, respectively. The data for the DapDc1 and DapDC2 crystals were collected in 0.5 steps over one 180 pass with exposure times of 1 and 0.5 s, respectively. Diffraction images were indexed and integrated using XDS (Kabsch, 2010). Scaling and data reduction were then performed using SCALA (Evans, 2006) and AIMLESS from the CCP4 suite (Winn et al., 2011). All relevant data-collection and processing parameters are given in Table 1.
3. Results and discussion The plant DapDc isoforms DapDc1 (coded by At3g14390) and DapDc2 (coded by At5g11880) were purified to homogeneity using Ni2+-affinity chromatography and size-exclusion chromatography. From a 1 l bacterial cell culture, 40 mg of protein was obtained.
Figure 3 Crystals of DapDc1 (left) and DapDc2 (right). The scale bar indicates 0.1 mm.
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crystallization communications Analysis of the proteins by SDS–PAGE and Coomassie Brilliant Blue staining showed a purity of at least 95% (Fig. 4). A multiple sequence alignment was carried out between the two homologues of diaminopimelate decarboxylase from A. thaliana (DapDc1 and DapDc2), eight additional bacterial species and a single archaeal species. The sequence alignment, shown in Fig. 2, illustrates the residues that are conserved between species, which are likely to Table 1 X-ray data-collection statistics for the two isoforms of plant diaminopimelate decarboxylase DapDc1 and DapDc2. Values in parentheses are for the highest resolution shell. The Matthews coefficients and solvent contents are based on two monomers.
˚) Wavelength (A No. of images Oscillation ( ) Space group ˚ , ) Unit-cell parameters (A ˚) Resolution (A Observed reflections Unique reflections Completeness (%) Rmerge† Rr.i.m.‡ Rp.i.m.§ Mean I/(I) Multiplicity ˚ 2) Wilson B value (A Molecules per asymmetric unit ˚ 3 Da1) VM (A Estimated solvent content (%)
DapDc1
DapDc2
0.95369 360 0.5 P21212 a = 80.9, b = 121.5, c = 88.6, = = = 90 48.62–2.00 (2.11–2.00) 440743 (64183) 59879 (8647) 100.00 (100.00) 0.129 (0.673) 0.138 (0.724) 0.051 (0.265) 11.3 (2.9) 7.4 (7.4) 26.4 2 2.22 45
0.95369 360 0.5 P21 a = 51.3, b = 107.9, c = 80.8, = = 90, = 98.6 46.02–2.27 (2.35–2.27) 151241 (13677) 39937 (3640) 99.8 (98.1) 0.155 (0.650) 0.213 (0.889) 0.145 (0.604) 6.9 (2.0) 3.8 (3.8) 26.1 2 2.34 48
P P P P †PRmerge = ‡ Rr.i.m. = hkl i jIi ðhklÞ hIðhklÞij=P hkl P i Ii ðhklÞ. 1=2 P 1g = i jIi ðhklÞ hIðhklÞij= Phkl fNðhklÞ=½NðhklÞ1=2 P P hkl i Ii ðhklÞ. § Rp.i.m. P hkl f1=½NðhklÞ 1g i jIi ðhklÞ hIðhklÞij= hkl i Ii ðhklÞ, where Ii(hkl) is the ith intensity measurement of reflection hkl, hI(hkl)i is its average and N(hkl) is the redundancy of a given reflection.
Figure 4 Purification of the recombinant plant diaminopimelate decarboxylase isoforms DapDc1 and DapDc2. (a) SDS–PAGE gel showing purification of the DapDc1 isoform. Lane 1, molecular-weight markers (labelled in kDa); lane 2, crude cell lysate; lane 3, pooled fractions after immobilized metal-affinity chromatography; lane 4, post size-exclusion chromatography. (b) SDS–PAGE gel showing purification of the DapDc2 isoform. Lane 1, crude cell lysate; lane 2, pooled fractions after immobilized metal-affinity chromatography; lane 3, post sizeexclusion chromatography; lane 4, molecular-weight markers (labelled in kDa).
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be important for protein function. Two consensus motifs for PLP binding have previously been identified in diaminopimelate decarboxylase from A. thaliana (Kim, 2006). The first motif includes a conserved lysine residue, Lys67 (amino-acid numbering corresponds to that DapDc1), which forms a Schiff base with the aldehyde moiety of PLP (Gokulan et al., 2003). The second motif features three consecutive glycine residues, Gly244–Gly246, which are hypothesized to interact with the phosphate moiety of the bound cofactor (Momany et al., 1995). Substrate binding is mediated via a salt bridge between the substrate and arginine residue Arg321 (Ray et al., 2002). A second arginine, Arg285, is also within bonding distance. Both of these arginines are conserved across all of the aligned species. Furthermore, two residues, Cys353 and Glu282, which are hypothesized to a play a catalytic role are both conserved (Jackson et al., 2000). The conservation of these residues within the active site suggest that the plant homologues of diaminopimelate decarboxylase utilize the same active-site architecture as prokaryotic homologues of the enzyme to achieve substrate and reaction specificity. The PACT screen conditions D10 and H2 produced DapDc1 crystals and condition F7 produced DapDc2 crystals. In-house optimization resulted in larger sized crystals (see xx2.3.1 and 2.3.2 for details). The best diffracting crystals of DapDc1 and DapDC2 were needle-shaped crystals of lengths 0.1 and 0.05 mm, respectively, that ˚ grew within 4 d at 293 K (Fig. 3). Complete data sets to 2.00 A ˚ resolution for DapDc1 and to 2.27 A resolution for DapDc2 were collected from these crystals. Data-collection and processing statistics are summarized in Table 1. Preliminary diffraction data analysis using XDS indicated that the DapDc1 and DapDc2 crystals belonged to space groups P21212 and P21, respectively. Initial phases were estimated by molecular replacement using MOLREP and Phaser from the CCP4 suite. The monomer of A. aeolicus DapDc, which shares the highest sequence identity with both isoforms of the plant DapDc (37%), was used as the search model (PDB entry 2p3e; Southeast Collaboratory for Structural Genomics/RIKEN Structural Genomics/Proteomics Initiative, unpublished work). The initial round of refinement after molecular replacement resulted in a decrease in the R factor from 0.5448 to 0.4739 and in Rfree from 0.5499 to 0.5215 for DapDc1 and a decrease in the R factor from 0.4889 to 0.4050 and in Rfree from 0.4804 to 0.4512 for DapDc2. In each case the majority of the model fits the electron density well, which confirms solution of the crystal structure by molecular replacement, while a few regions, such as the terminal regions, do not fit so well. This results in relatively high Rfree values, but we are continuing to refine these models. The analysis suggested two DapDc1 molecules per asymmetric unit with a Matthews ˚ 3 Da1 and a solvent content coefficient (Matthews, 1968) of 2.22 A of 45% and two DapDc2 molecules per asymmetric unit with a ˚ 3 Da1 and a solvent Matthews coefficient (Matthews, 1968) of 2.34 A content of 48%. These findings suggest that DapDc1 and DapDc2 may exist as dimers. This is in agreement with other studies that suggest that the diaminopimelate decarboxylases from M. jannaschii (Ray et al., 2002), H. pylori (Hu et al., 2008) and M. tuberculosis (Gokulan et al., 2003) also form dimers. The structures of the two isomers of plant DapDc will be published elsewhere. In summary, we present the purification, crystallization and preliminary X-ray diffraction analysis of two DapDc isomers from A. thaliana, the first eukaryotic DapDc homologue studied thus far. The structural information gathered from the characterization of plant DapDc may be used to identify specific differences between plant and bacterial homologues of this lysine-biosynthetic enzyme. This information could be useful in the design of herbicide inhibitors that selectively target plant DapDc. Oliver et al.
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crystallization communications We acknowledge the support and assistance of the friendly staff at the CSIRO Collaborative Crystallization Centre at CSIRO Material Science and Engineering, Parkville, Melbourne and the MX beamline scientists at the Australian Synchrotron, Victoria, Australia. Parts of this research were undertaken at the MX2 beamline of the Australian Synchrotron, Victoria, Australia. Travel to the Australian Synchrotron was supported by the New Zealand Synchrotron Group. HS acknowledges the 2012 Researcher Exchange Program between JSPS and RSNZ. RCJD acknowledges (i) the New Zealand Royal Society Marsden Fund for funding support, in part (contract UOC1013), and (ii) the US Army Research Laboratory and US Army Research Office under contract/grant No. W911NF-11-1-0481 for support, in part. AOH acknowledges a National Science Foundation (NSF) award (MCB-#1120541) and a Rochester Institute of Technology (RIT) College of Science 2012 Dean’s Research Initiation Grant. MML was supported by the NSF award to AOH. MDWG is the recipient of the C. R. Roper Fellowship. We especially thank Jackie Healy for her technical support.
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