The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 ( unit-cell parameters a = b = 136.83, c = 99.82 angstrom, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 angstrom resolution using the laboratory X-ray source and are suitable for crystal structure determination

Forwood, J. K.

Hume, D. A.

Kobe, B.

Martin, J. L.

Serek, R.

PubMed

The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 A, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 A resolution using the laboratory X-ray source and are suitable for crystal structure determination

Serek, Robert

Forwood, Jade K

Hume, David A

Martin, Jennifer L

Kobe, Bostjan

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Crystallization of the C-terminal domain of the mouse braincytosolic long-chain acyl-CoA thioesteraseCitation for published version:Serek, R, Forwood, JK, Hume, DA, Martin, JL & Kobe, B 2006, 'Crystallization of the C-terminal domain ofthe mouse brain cytosolic long-chain acyl-CoA thioesterase' Acta Crystallographica Section F StructuralBiology and Crystallization Communications, vol 62, no. Pt 2, pp. 133-5., 10.1107/S1744309106000030Digital Object Identifier (DOI):10.1107/S1744309106000030Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher final version (usually the publisher pdf)Published In:Acta Crystallographica Section F Structural Biology and Crystallization CommunicationsPublisher Rights Statement:Copyright 2006 International Union of CrystallographyGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 20. Feb. 2015crystallization communicationsActa Cryst. (2006). F62, 133–135 doi:10.1107/S1744309106000030 133Acta Crystallographica Section FStructural Biologyand CrystallizationCommunicationsISSN 1744-3091Crystallization of the C-terminal domain of themouse brain cytosolic long-chain acyl-CoAthioesteraseRobert Serek,a Jade K. Forwood,aDavid A. Hume,a,b,c,d Jennifer L.Martina,b,d and Bostjan Kobea,b,d*aSchool of Molecular and Microbial Sciences,University of Queensland, Brisbane,Queensland 4072, Australia, bInstitute forMolecular Bioscience, University ofQueensland, Brisbane, Queensland 4072,Australia, cCooperative Research Centre forChronic Inflammatory Diseases, University ofQueensland, Brisbane, Queensland 4072,Australia, and dSpecial Research Centre forFunctional and Applied Genomics, University ofQueensland, Brisbane, Queensland 4072,AustraliaCorrespondence e-mail: b.kobe@uq.edu.auReceived 25 November 2005Accepted 3 January 2006The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses thehydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT(4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of themouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteriaand crystallized. The crystals were obtained by vapour diffusion using PEG 2000MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry ofspace group R32 (unit-cell parameters a = b = 136.83, c= 99.82 A˚,  = 120). Twomolecules are expected in the asymmetric unit. The crystals diffract to 2.4 A˚resolution using the laboratory X-ray source and are suitable for crystalstructure determination.1. IntroductionLong-chain acyl-CoAs are intermediates in lipid metabolism andregulators of cellular processes including ion transport, vesicle traf-ficking, protein phosphorylation and gene expression (Faergeman &Knudsen, 1997; Hunt & Alexson, 2002; Yamada, 2005). Intracellularlevels of acyl-CoAs are controlled by the ratio of synthesizing andhydrolysing activities. Acyl-CoA thioesterases (also known as acyl-CoA hydrolases; EC 3.1.2.1 and 3.1.2.2) catalyse the hydrolysis ofacyl-CoAs to free fatty acids and CoA-SH. These enzymes are widelydistributed among different tissues in mammals (Hunt & Alexson,2002).High levels of long-chain acyl-CoA hydrolysing activity (EC3.1.2.2) have been detected in mouse and human brain (Anderson &Erwin, 1971; Yamada, 2005). Full-length cDNAs corresponding to sixdifferent isoforms were cloned from the human brain and shown tobe derived from a single gene (Hunt et al., 2005; Yamada et al., 2002).When expressed in bacteria, four of the six isoforms showed acyl-CoA hydrolase activity, while two C-terminally truncated isoformsdid not (Yamada et al., 2002). Because several different groups haveindependently cloned the enzyme, several different names exist,including BACH (Kuramochi et al., 2002; Yamada et al., 1997, 1999),CTE-II (Engberg et al., 1997) and ACT (Broustas et al., 1996). Therecently suggested revised nomenclature designates this protein asAcot7 (Hunt et al., 2005). The mouse brain contains a 43 kDa Acot7as the major isoform and other lesser isoforms including a 50 kDaisoform (Takagi et al., 2004). The enzyme is specific for acyl-CoAhydrolysis, but has a broad chain-length specificity, hydrolysing acyl-CoAs with carbon numbers C6–C20 (Broustas & Hajra, 1995;Yamada et al., 1994, 1996, 1999, 2002).Acot7 contains two copies of the 4HBT domain (named after4-hydroxybenzoyl-CoA thioesterase). The structure of 4HBT fromPseudomonas revealed a ‘Hotdog’ fold comprising a -sheet‘bun’ wrapped around an -helical ‘sausage’ (Benning et al., 1998).This fold was first observed in the structure of Escherichia coli-hydroxydecanoyl thiol ester dehydratase (FabA; Leesong et al.,1996) and has since been found in a number of apparently unrelatedproteins (Dillon & Bateman, 2004). The acyl-CoA thioesterases forma distinct class of Hotdog-fold proteins with little available structuralinformation.To shed light on the molecular function of long-chain acyl-CoAthioesterases, including the enzymatic mechanism and the specificity,we set out to determine the three-dimensional structure of mouse# 2006 International Union of CrystallographyAll rights reservedAcot7. As the first step towards this goal, we report here thecrystallization and preliminary X-ray diffraction analysis of theC-terminal 4HBT domain of this protein.2. Experimental methods2.1. Expression and purificationThe DNA encoding amino acids 160–338 of variant 1 of mouse(Mus musculus) Acot7 (GeneInfo Identification No. 19923052;Kuramochi et al., 2002) was amplified by PCR using cDNA preparedfrom LPS-stimulated mouse macrophage cells as a template (Wells etal., 2003). The reactions were catalysed by Triplemaster proofreading,blunt-ended polymerase mix (Eppendorf), using gene-specificprimers at a concentration of 10 ng ml1. The PCR conditions wereinitial denaturation at 369 K for 4 min, followed by 25 cycles of 369 Kfor 30 s, 328 K for 30 s and 345 K for 1 min, and finally one cycle of345 K for 5 min. The PCR product was cloned into the Gateway entryvector pENTR-D-TOPO (Invitrogen) following the manufacturer’sinstructions and transformed into chemically competent Top10 cells(Invitrogen) by heat-shock. Colonies were inoculated in 4 ml LBmedium in 15 ml Falcon tubes and plasmid DNA was purified fromthese cultures using a DNA-extraction kit (Roche). Genes clonedinto pENTR-D-TOPO were recombined into the expression vectorpDEST-17 (Invitrogen) by the Gateway LR reaction following themanufacturer’s instructions; this vector allows expression of a fusionprotein containing an N-terminal hexahistidine tag (His tag). Theresulting entry and expression vectors were initially assessed bydigestion with the restriction enzymes NotI (entry vector) andHindIII and BamHI (expression vector) and confirmed by DNAsequencing.The expression vector was used to transform the chemicallycompetent E. coli strain BL21(DE3)pLysS by heat-shock. A singlecolony was inoculated into 20 ml Luria–Bertani (LB) medium andgrown overnight at 310 K in the presence of ampicillin and then usedto inoculate 2 l LB containing ampicillin. The culture was grownaerobically at 310 K until the OD600nm reached 1 and induced witheither 1 mM IPTG or 10–30 mM lactose. The temperature wasreduced to room temperature upon induction and the culture wasgrown for a further 12 h. At harvest, the OD600 nm was typically 5–6and the yield of recombinant Acot7 was 7–8 mg per litre of culture.The His-tagged protein was purified by immobilized metal-affinitychromatography (IMAC; nickel-affinity gel, Sigma). After elution,the buffer was exchanged using a PD-10 desalting column (BioRad)with 200 mM Tris pH 8.5, 150 mM NaCl, 1 mM DTT and the proteinwas diluted to3.5 mg ml1 for crystallizaton. The protein was >95%pure as assessed by SDS–PAGE.2.2. CrystallizationPrior to crystallization, the protein sample was supplemented with0.25 mg ml1 coenzyme A (CoA). Crystallization conditions werescreened by the sparse-matrix approach using the hanging-dropvapour-diffusion technique (Jancarik & Kim, 1991; McPherson,1982). 1 ml protein solution was combined with 1 ml reservoir solutionand suspended over 0.1 ml reservoir solution. Small crystals wereinitially observed in 25% polyethylene glycol (PEG) 3350, 0.1 M TrispH 5.5, 0.2M ammonium acetate. Crystals could be grown in the pHrange 5.5–8, with the best crystals obtained in 20–30% of either PEG3350, 0.1M Tris pH 7–8 or 20% PEG 2000 monomethylether (MME),0.1 M Tris pH 7.0. SDS–PAGE of dissolved crystals confirmed thatthey were formed by the Acot7 fragment.2.3. Diffraction data collectionFor X-ray diffraction experiments, crystals were transiently soakedin a solution corresponding to the reservoir solution but supple-mented with 15% glycerol and were cooled at 100 K in a nitrogenstream (Cryocool, Cryo Industries, New Hampshire, USA). Datawere collected from single crystals using an R-AXIS IV++ image-platedetector and Cu K radiation from a Rigaku FR-E rotating-anodegenerator (Rigaku/MSC, Texas, USA). Data were autoindexed andprocessed with the program CrystalClear (Rigaku/MSC; Table 1). Arepresentative oscillation image is shown in Fig. 1.3. Results and discussionWe obtained crystals of the protein corresponding to the C-terminal4HBT domain of the mouse long-chain acyl-CoA thioesterasecrystallization communications134 Serek et al.  Long-chain acyl-CoA thioesterase Acta Cryst. (2006). F62, 133–135Table 1Diffraction data-collection statistics.Values in parentheses are for the highest resolution shell.Reservoir solution 0.1 M Tris, 20–30% PEG 2000 MME pH 7.0Space group R32Unit-cell parametersa = b (A˚) 136.83c (A˚) 99.82 () 120Resolution range (A˚) 30.0–2.4 (2.48–2.40)Observations 60405Unique reflections 14175Average redundancy 4.26 (4.19)Completeness (%) 99.9 (99.6)Rmerge 0.05 (0.56)Average I/(I) 12.7 (2.2)† Rmerge =Phkl ½PiðjIhkl;i  hIhklijÞ=Phkl;ihIhkli, where Ihkl,i is the intensity of anindividual measurement of the reflection with Miller indices h, k and l and hIhkli is themean intensity of that reflection for I > 3(I).Figure 1Oscillation image (1) of the crystal described in Table 1. The crystal-to-detectordistance is 120 mm; the resolution at the edge of the image is 2.0 A˚.(Acot7/mBACH) using either PEG 3350 or PEG 2000 MME as theprecipitant. The crystals are rod-shaped (dimensions 0.2  0.2 0.1 mm) and have the symmetry of space group R32. The crystalsappear after a few days and grow to maximum dimensions withinthree weeks. There are likely to be two molecules of the protein in theasymmetric unit [assuming two molecules, the Matthews coefficientVM (Matthews, 1968) and the solvent content are 2.0 A˚3 Da1 and38%, respectively]. The crystals diffract X-rays using the laboratorysource to 2.4 A˚ resolution. The data-collection statistics are shown inTable 1.Size-exclusion chromatography suggests the protein expressedfrom our construct exists in a dimeric form (data not shown), which isconsistent with known 4HBT domain structures (Dillon & Bateman,2004). Likewise, the 43 kDa isoform showed a molecular weight of100 kDa using gel filtration (Yamada et al., 1996, 1999).Determination of the crystal structure of this protein will provideimportant insights into the catalytic mechanism and regulation of themammalian long-chain acyl-CoA thioesterases. The full-lengthprotein contains two 4HBT domains fused as part of the samepolypeptide (the N- and C-terminal domains share 27% sequenceidentity based on alignment of residues 18–171 and 180–330,respectively); it is currently unknown what the distinct activities ofthe two domains are and how they cooperate within the full-lengthproteins. Based on a spectrophotometric assay (Yamada et al., 1994),our construct of the C-terminal 4HBT domain shows some palmitoyl-CoA hydrolizing activity (J. K. Forwood, W. N. Meng, R. Serek andB. Kobe, unpublished results). While both 4HBT domains arepredicted to have the structure corresponding to the Hotdog fold, nostructures are currently available of any eukaryotic proteinscontaining this structural motif.We thank Pawel Listwan for help with cloning and for criticallyreading the manuscript. This work was supported by the AustralianResearch Council (ARC; to JLM and BK); BK is a National Healthand Medical Research Council of Australia (NHMRC) ResearchFellow and ARC Federation Fellow.ReferencesAnderson, A. D. & Erwin, V. G. (1971). J. Neurochem. 18, 1179–1186.Benning, M. M., Wesenberg, G., Liu, R., Taylor, K. L., Dunaway-Mariano, D.& Holden, H. M. (1998). J. Biol. Chem. 273, 33572–33579.Broustas, C. G. & Hajra, A. K. (1995). J. Neurochem. 64, 2345–2353.Broustas, C. G., Larkins, L. K., Uhler, M. D. & Hajra, A. K. (1996). J. Biol.Chem. 271, 10470–10476.Dillon, S. C. & Bateman, A. (2004). BMC Bioinformatics, 5, 109.Engberg, S. T., Aoyama, T., Alexson, S. E., Hashimoto, T. & Svensson, L. T.(1997). Biochem. J. 323, 525–531.Faergeman, N. J. & Knudsen, J. (1997). Biochem J. 323, 1–12.Hunt, M. C. & Alexson, S. E. (2002). Prog. Lipid Res. 41, 99–130.Hunt, M. C., Yamada, J., Maltais, L. J., Wright, M. W., Podesta, E. J. & Alexson,S. E. (2005). J. Lipid Res. 46, 2029–2032.Jancarik, J. & Kim, S.-H. (1991). J. Appl. Cryst. 24, 409–411.Kuramochi, Y., Takagi-Sakuma, M., Kitahara, M., Emori, R., Asaba, Y.,Sakaguchi, R., Watanabe, T., Kuroda, J., Hiratsuka, K., Nagae, Y., Suga, T. &Yamada, J. (2002). Brain Res. Mol. Brain Res. 98, 81–92.Leesong, M., Henderson, B. S., Gillig, J. R., Schwab, J. M. & Smith, J. L. (1996).Structure, 4, 253–264.McPherson, A. (1982). Preparation and Analysis of Protein Crystals. NewYork: John Wiley & Sons.Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.Takagi, M., Kawabe, K., Suga, T. & Yamada, J. (2004). Arch. Biochem.Biophys. 429, 100–105.Wells, C. A., Ravasi, T., Faulkner, G. J., Carninci, P., Okazaki, Y., Hayashizaki,Y., Sweet, M., Wainwright, B. J. & Hume, D. A. (2003). BMC Immunol. 4, 5.Yamada, J. (2005). Amino Acids, 28, 273–278.Yamada, J., Furihata, T., Iida, N., Watanabe, T., Hosokawa, M., Satoh, T.,Someya, A., Nagaoka, I. & Suga, T. (1997). Biochem. Biophys. Res.Commun. 232, 198–203.Yamada, J., Furihata, T., Tamura, H., Watanabe, T. & Suga, T. (1996). Arch.Biochem. Biophys. 326, 106–114.Yamada, J., Kuramochi, Y., Takagi, M., Watanabe, T. & Suga, T. (2002).Biochem. Biophys. Res. Commun. 299, 49–56.Yamada, J., Kurata, A., Hirata, M., Taniguchi, T., Takama, H., Furihata, T.,Shiratori, K., Iida, N., Takagi-Sakuma, M., Watanabe, T., Kurosaki, K.,Endo, T. & Suga, T. (1999). J. Biochem. (Tokyo), 126, 1013–1019.Yamada, J., Matsumoto, I., Furihata, T., Sakuma, M. & Suga, T. (1994). Arch.Biochem. Biophys. 308, 118–125.crystallization communicationsActa Cryst. (2006). F62, 133–135 Serek et al.  Long-chain acyl-CoA thioesterase 135

English

308, 118–125. crystallization communications Acta Cryst.

Brain Res.

Preparation and Analysis of Protein Crystals.

Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

Robert Serek

Jade K. Forwood

David A. Hume

Jennifer L. Martin

Bostjan Kobe

Crossref

University of Queensland eSpace

crystallization communications
Acta Cryst. (2006). F62, 133–135 doi:10.1107/S1744309106000030 133
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
Crystallization of the C-terminal domain of the
mouse brain cytosolic long-chain acyl-CoA
thioesterase
Robert Serek,a Jade K. Forwood,a
David A. Hume,a,b,c,d Jennifer L.
Martina,b,d and Bostjan Kobea,b,d*
aSchool of Molecular and Microbial Sciences,
University of Queensland, Brisbane,
Queensland 4072, Australia, bInstitute for
Molecular Bioscience, University of
Queensland, Brisbane, Queensland 4072,
Australia, cCooperative Research Centre for
Chronic Inflammatory Diseases, University of
Queensland, Brisbane, Queensland 4072,
Australia, and dSpecial Research Centre for
Functional and Applied Genomics, University of
Queensland, Brisbane, Queensland 4072,
Australia
Correspondence e-mail: b.kobe@uq.edu.au
Received 25 November 2005
Accepted 3 January 2006
The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the
hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT
(4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the
mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria
and crystallized. The crystals were obtained by vapour diffusion using PEG 2000
MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of
space group R32 (unit-cell parameters a = b = 136.83, c= 99.82 A˚,  = 120). Two
molecules are expected in the asymmetric unit. The crystals diffract to 2.4 A˚
resolution using the laboratory X-ray source and are suitable for crystal
structure determination.
1. Introduction
Long-chain acyl-CoAs are intermediates in lipid metabolism and
regulators of cellular processes including ion transport, vesicle traf-
ficking, protein phosphorylation and gene expression (Faergeman &
Knudsen, 1997; Hunt & Alexson, 2002; Yamada, 2005). Intracellular
levels of acyl-CoAs are controlled by the ratio of synthesizing and
hydrolysing activities. Acyl-CoA thioesterases (also known as acyl-
CoA hydrolases; EC 3.1.2.1 and 3.1.2.2) catalyse the hydrolysis of
acyl-CoAs to free fatty acids and CoA-SH. These enzymes are widely
distributed among different tissues in mammals (Hunt & Alexson,
2002).
High levels of long-chain acyl-CoA hydrolysing activity (EC
3.1.2.2) have been detected in mouse and human brain (Anderson &
Erwin, 1971; Yamada, 2005). Full-length cDNAs corresponding to six
different isoforms were cloned from the human brain and shown to
be derived from a single gene (Hunt et al., 2005; Yamada et al., 2002).
When expressed in bacteria, four of the six isoforms showed acyl-
CoA hydrolase activity, while two C-terminally truncated isoforms
did not (Yamada et al., 2002). Because several different groups have
independently cloned the enzyme, several different names exist,
including BACH (Kuramochi et al., 2002; Yamada et al., 1997, 1999),
CTE-II (Engberg et al., 1997) and ACT (Broustas et al., 1996). The
recently suggested revised nomenclature designates this protein as
Acot7 (Hunt et al., 2005). The mouse brain contains a 43 kDa Acot7
as the major isoform and other lesser isoforms including a 50 kDa
isoform (Takagi et al., 2004). The enzyme is specific for acyl-CoA
hydrolysis, but has a broad chain-length specificity, hydrolysing acyl-
CoAs with carbon numbers C6–C20 (Broustas & Hajra, 1995;
Yamada et al., 1994, 1996, 1999, 2002).
Acot7 contains two copies of the 4HBT domain (named after
4-hydroxybenzoyl-CoA thioesterase). The structure of 4HBT from
Pseudomonas revealed a ‘Hotdog’ fold comprising a -sheet
‘bun’ wrapped around an -helical ‘sausage’ (Benning et al., 1998).
This fold was first observed in the structure of Escherichia coli
-hydroxydecanoyl thiol ester dehydratase (FabA; Leesong et al.,
1996) and has since been found in a number of apparently unrelated
proteins (Dillon & Bateman, 2004). The acyl-CoA thioesterases form
a distinct class of Hotdog-fold proteins with little available structural
information.
To shed light on the molecular function of long-chain acyl-CoA
thioesterases, including the enzymatic mechanism and the specificity,
we set out to determine the three-dimensional structure of mouse
# 2006 International Union of Crystallography
All rights reserved
Acot7. As the first step towards this goal, we report here the
crystallization and preliminary X-ray diffraction analysis of the
C-terminal 4HBT domain of this protein.
2. Experimental methods
2.1. Expression and purification
The DNA encoding amino acids 160–338 of variant 1 of mouse
(Mus musculus) Acot7 (GeneInfo Identification No. 19923052;
Kuramochi et al., 2002) was amplified by PCR using cDNA prepared
from LPS-stimulated mouse macrophage cells as a template (Wells et
al., 2003). The reactions were catalysed by Triplemaster proofreading,
blunt-ended polymerase mix (Eppendorf), using gene-specific
primers at a concentration of 10 ng ml1. The PCR conditions were
initial denaturation at 369 K for 4 min, followed by 25 cycles of 369 K
for 30 s, 328 K for 30 s and 345 K for 1 min, and finally one cycle of
345 K for 5 min. The PCR product was cloned into the Gateway entry
vector pENTR-D-TOPO (Invitrogen) following the manufacturer’s
instructions and transformed into chemically competent Top10 cells
(Invitrogen) by heat-shock. Colonies were inoculated in 4 ml LB
medium in 15 ml Falcon tubes and plasmid DNA was purified from
these cultures using a DNA-extraction kit (Roche). Genes cloned
into pENTR-D-TOPO were recombined into the expression vector
pDEST-17 (Invitrogen) by the Gateway LR reaction following the
manufacturer’s instructions; this vector allows expression of a fusion
protein containing an N-terminal hexahistidine tag (His tag). The
resulting entry and expression vectors were initially assessed by
digestion with the restriction enzymes NotI (entry vector) and
HindIII and BamHI (expression vector) and confirmed by DNA
sequencing.
The expression vector was used to transform the chemically
competent E. coli strain BL21(DE3)pLysS by heat-shock. A single
colony was inoculated into 20 ml Luria–Bertani (LB) medium and
grown overnight at 310 K in the presence of ampicillin and then used
to inoculate 2 l LB containing ampicillin. The culture was grown
aerobically at 310 K until the OD600nm reached 1 and induced with
either 1 mM IPTG or 10–30 mM lactose. The temperature was
reduced to room temperature upon induction and the culture was
grown for a further 12 h. At harvest, the OD600 nm was typically 5–6
and the yield of recombinant Acot7 was 7–8 mg per litre of culture.
The His-tagged protein was purified by immobilized metal-affinity
chromatography (IMAC; nickel-affinity gel, Sigma). After elution,
the buffer was exchanged using a PD-10 desalting column (BioRad)
with 200 mM Tris pH 8.5, 150 mM NaCl, 1 mM DTT and the protein
was diluted to3.5 mg ml1 for crystallizaton. The protein was >95%
pure as assessed by SDS–PAGE.
2.2. Crystallization
Prior to crystallization, the protein sample was supplemented with
0.25 mg ml1 coenzyme A (CoA). Crystallization conditions were
screened by the sparse-matrix approach using the hanging-drop
vapour-diffusion technique (Jancarik & Kim, 1991; McPherson,
1982). 1 ml protein solution was combined with 1 ml reservoir solution
and suspended over 0.1 ml reservoir solution. Small crystals were
initially observed in 25% polyethylene glycol (PEG) 3350, 0.1 M Tris
pH 5.5, 0.2M ammonium acetate. Crystals could be grown in the pH
range 5.5–8, with the best crystals obtained in 20–30% of either PEG
3350, 0.1M Tris pH 7–8 or 20% PEG 2000 monomethylether (MME),
0.1 M Tris pH 7.0. SDS–PAGE of dissolved crystals confirmed that
they were formed by the Acot7 fragment.
2.3. Diffraction data collection
For X-ray diffraction experiments, crystals were transiently soaked
in a solution corresponding to the reservoir solution but supple-
mented with 15% glycerol and were cooled at 100 K in a nitrogen
stream (Cryocool, Cryo Industries, New Hampshire, USA). Data
were collected from single crystals using an R-AXIS IV++ image-plate
detector and Cu K radiation from a Rigaku FR-E rotating-anode
generator (Rigaku/MSC, Texas, USA). Data were autoindexed and
processed with the program CrystalClear (Rigaku/MSC; Table 1). A
representative oscillation image is shown in Fig. 1.
3. Results and discussion
We obtained crystals of the protein corresponding to the C-terminal
4HBT domain of the mouse long-chain acyl-CoA thioesterase
crystallization communications
134 Serek et al.  Long-chain acyl-CoA thioesterase Acta Cryst. (2006). F62, 133–135
Table 1
Diffraction data-collection statistics.
Values in parentheses are for the highest resolution shell.
Reservoir solution 0.1 M Tris, 20–30% PEG 2000 MME pH 7.0
Space group R32
Unit-cell parameters
a = b (A˚) 136.83
c (A˚) 99.82
 () 120
Resolution range (A˚) 30.0–2.4 (2.48–2.40)
Observations 60405
Unique reflections 14175
Average redundancy 4.26 (4.19)
Completeness (%) 99.9 (99.6)
Rmerge 0.05 (0.56)
Average I/(I) 12.7 (2.2)
† Rmerge =
P
hkl ½
P
iðjIhkl;i  hIhklijÞ=
P
hkl;ihIhkli, where Ihkl,i is the intensity of an
individual measurement of the reflection with Miller indices h, k and l and hIhkli is the
mean intensity of that reflection for I > 3(I).
Figure 1
Oscillation image (1) of the crystal described in Table 1. The crystal-to-detector
distance is 120 mm; the resolution at the edge of the image is 2.0 A˚.
(Acot7/mBACH) using either PEG 3350 or PEG 2000 MME as the
precipitant. The crystals are rod-shaped (dimensions 0.2  0.2 
0.1 mm) and have the symmetry of space group R32. The crystals
appear after a few days and grow to maximum dimensions within
three weeks. There are likely to be two molecules of the protein in the
asymmetric unit [assuming two molecules, the Matthews coefficient
VM (Matthews, 1968) and the solvent content are 2.0 A˚
3 Da1 and
38%, respectively]. The crystals diffract X-rays using the laboratory
source to 2.4 A˚ resolution. The data-collection statistics are shown in
Table 1.
Size-exclusion chromatography suggests the protein expressed
from our construct exists in a dimeric form (data not shown), which is
consistent with known 4HBT domain structures (Dillon & Bateman,
2004). Likewise, the 43 kDa isoform showed a molecular weight of
100 kDa using gel filtration (Yamada et al., 1996, 1999).
Determination of the crystal structure of this protein will provide
important insights into the catalytic mechanism and regulation of the
mammalian long-chain acyl-CoA thioesterases. The full-length
protein contains two 4HBT domains fused as part of the same
polypeptide (the N- and C-terminal domains share 27% sequence
identity based on alignment of residues 18–171 and 180–330,
respectively); it is currently unknown what the distinct activities of
the two domains are and how they cooperate within the full-length
proteins. Based on a spectrophotometric assay (Yamada et al., 1994),
our construct of the C-terminal 4HBT domain shows some palmitoyl-
CoA hydrolizing activity (J. K. Forwood, W. N. Meng, R. Serek and
B. Kobe, unpublished results). While both 4HBT domains are
predicted to have the structure corresponding to the Hotdog fold, no
structures are currently available of any eukaryotic proteins
containing this structural motif.
We thank Pawel Listwan for help with cloning and for critically
reading the manuscript. This work was supported by the Australian
Research Council (ARC; to JLM and BK); BK is a National Health
and Medical Research Council of Australia (NHMRC) Research
Fellow and ARC Federation Fellow.
References
Anderson, A. D. & Erwin, V. G. (1971). J. Neurochem. 18, 1179–1186.
Benning, M. M., Wesenberg, G., Liu, R., Taylor, K. L., Dunaway-Mariano, D.
& Holden, H. M. (1998). J. Biol. Chem. 273, 33572–33579.
Broustas, C. G. & Hajra, A. K. (1995). J. Neurochem. 64, 2345–2353.
Broustas, C. G., Larkins, L. K., Uhler, M. D. & Hajra, A. K. (1996). J. Biol.
Chem. 271, 10470–10476.
Dillon, S. C. & Bateman, A. (2004). BMC Bioinformatics, 5, 109.
Engberg, S. T., Aoyama, T., Alexson, S. E., Hashimoto, T. & Svensson, L. T.
(1997). Biochem. J. 323, 525–531.
Faergeman, N. J. & Knudsen, J. (1997). Biochem J. 323, 1–12.
Hunt, M. C. & Alexson, S. E. (2002). Prog. Lipid Res. 41, 99–130.
Hunt, M. C., Yamada, J., Maltais, L. J., Wright, M. W., Podesta, E. J. & Alexson,
S. E. (2005). J. Lipid Res. 46, 2029–2032.
Jancarik, J. & Kim, S.-H. (1991). J. Appl. Cryst. 24, 409–411.
Kuramochi, Y., Takagi-Sakuma, M., Kitahara, M., Emori, R., Asaba, Y.,
Sakaguchi, R., Watanabe, T., Kuroda, J., Hiratsuka, K., Nagae, Y., Suga, T. &
Yamada, J. (2002). Brain Res. Mol. Brain Res. 98, 81–92.
Leesong, M., Henderson, B. S., Gillig, J. R., Schwab, J. M. & Smith, J. L. (1996).
Structure, 4, 253–264.
McPherson, A. (1982). Preparation and Analysis of Protein Crystals. New
York: John Wiley & Sons.
Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.
Takagi, M., Kawabe, K., Suga, T. & Yamada, J. (2004). Arch. Biochem.
Biophys. 429, 100–105.
Wells, C. A., Ravasi, T., Faulkner, G. J., Carninci, P., Okazaki, Y., Hayashizaki,
Y., Sweet, M., Wainwright, B. J. & Hume, D. A. (2003). BMC Immunol. 4, 5.
Yamada, J. (2005). Amino Acids, 28, 273–278.
Yamada, J., Furihata, T., Iida, N., Watanabe, T., Hosokawa, M., Satoh, T.,
Someya, A., Nagaoka, I. & Suga, T. (1997). Biochem. Biophys. Res.
Commun. 232, 198–203.
Yamada, J., Furihata, T., Tamura, H., Watanabe, T. & Suga, T. (1996). Arch.
Biochem. Biophys. 326, 106–114.
Yamada, J., Kuramochi, Y., Takagi, M., Watanabe, T. & Suga, T. (2002).
Biochem. Biophys. Res. Commun. 299, 49–56.
Yamada, J., Kurata, A., Hirata, M., Taniguchi, T., Takama, H., Furihata, T.,
Shiratori, K., Iida, N., Takagi-Sakuma, M., Watanabe, T., Kurosaki, K.,
Endo, T. & Suga, T. (1999). J. Biochem. (Tokyo), 126, 1013–1019.
Yamada, J., Matsumoto, I., Furihata, T., Sakuma, M. & Suga, T. (1994). Arch.
Biochem. Biophys. 308, 118–125.
crystallization communications
Acta Cryst. (2006). F62, 133–135 Serek et al.  Long-chain acyl-CoA thioesterase 135


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Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

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