The Expression, Purification and Crystallization of the E Subunit of the FI Portion of the ATPase of Escherichia coli Rachel Codd’, Graeme B. Cox’, J. Mitchell Guss”f, Robert G. Solomon2 and Dianne Webb’

The E subunit of the FOP,-XTPasr from Excherichia coli has been rxpressrd in ti:. co/i as a fusion protein with glutathione S-trattsfclrase from the parasit’ic hrlmint h Sch icstowlmrr ,jnpnicrrnl. The E subunit reltaased by t hrombin treatment of t,hr puritietl fusion protein carrietl two amino acid changes. Al(: anti 372s. and was 0l)taineti in a yirltl of ahout five milligrams per litrr of cwltured wlls. The two amino acid changes were shown not to affec~i function. The protein has hew cq-st.allized in a form suit,ahle for S-ray tiiffra~tion st ruvturc~ analysis. Thr crystals are hexagonal. space group I%,22 ( or I’A,%P). wit.h 0 = t) = !bW A. (‘ = 57.1 .i and ;’ = 120”. Thr cliffrac+iott from small cv-ystals extr~ntls to at least L’.!) .A rrsolutioti. hYr!/rrvrds;

ATT’ synthase:

i: subunit:

X-ray

I’rotott-trarlslo(~atiny FOF,~-ATPasrs art’ Ioc*atvtl in mitoc~~londrial, chloroplast untl I)ac4flrial ttwmbrants. 111 ILschPrichin wli the twzyrnr has two physiological roles. I’nder wnditions whew rrspitw t,ion can generate a proton gradient. thr twzytw caatalysw ATT’ synt)hesis (oxidatiw Ithosphor~li~tiott) anti t.hus is known as “ATT-’ synthast~“. At a low mrmhrane potential gratlient thfl rrtzytnr hydrolyses ATT’ and pumps protons in t ht, opposite, dirwt.iott. Therv are a number of t-went tw-itws on thr structuw anti tnechanistn of acation of FoF, ATPaw (e.g. Senior, 1990: Fillingatrtt~. 1990). The structure is highly cwnservetl and thtk wrnples cart I)r readily separated int)o two portions, the water soluble F,-ATPase and the rnemhranr bound FO portion which acts as the proton JKIW, The 15. co/i F1 -ATPaw ( WF, ATPawS) is comprised of five protein subunits (r. 8. y. S. E) in ortler of drcreasing size and F. of t,hrer subnnit,s (~1% b, c). The cvmposititrn of t htl F, port ion is thought t,o he rJ~,$r: ant1 the t Author to whom all c,orrrsl)ondrnc,r should tw addressed. $ Abbreviations used: lN'F,i\TI'asr, BwlrPrichiu co/i: Ii, ATPaw: MI’I). “-rnrt,hvl-“.4-I,~t~t,atl~(~iol: II’T(:. isopropyl-B-t)-thiogalactosidr: HPIAI. high-pressure liquid cbhromat,ograph,v: PAGE. polyac~rylarnide gel rlrct~rophorrsis; PEG. polyethylene gly~~l.

diffrac~tiotr:

secp“tl’Y’s

c-rystallization:

t~sprtwiott

of’ the inclividual polypfttitlw ot tG’F,ATPast~ ha\-ights) of’ the subunits arc: x. 513 (55,300): 8, 15!1 (.3).200): 7. PM (31 ,100): 6. Ii7 (19,300) ant! E. I38 (I5.000). The c subunit of thrl II:(‘F,XTPaw has twcw petrified and c*harac:trrizecl as a gtoltular prolvirl of’ molec~ular lvcight 1.i.OOO to 16.000 with H,II r-hvlical content of ahout 100,, (Sterttwvis Hr Smith. 1980). The ATPaw acstivitv of purifietl Ii‘, ATPaw wts inhibitcvl hy the adciition of purifit~tl c sttttuttit but the adclition of i: to t hr tnc,mttrari~-I,outitl FOP, ATPaw cwmplex (lid not inhibit ATPaw act ivit? (SternwGs & Smith. l!M)). I)unti vf trl. (l!)X7) concblrd that thr L subunit inhittit (4 t ht. H(‘F,ATPas;e activity tty wducittg t hr ratv of’ protluct release from t htl c3talytic sitv. ‘i’hc~ i: su It unit has RISO twrn shown to form a spcc*itic*. high affinity tyuitnolar cwmplcx with the 7 subunit and f: is thought to hint1 to thr I+‘- ATPast through intrrI 9x2). mu ac%iotis \vit h t.hr ;: sut)uttitj (I)utitt. E(‘F,ATI’ast~ tacking thta E subunit was ttttaltl(l tc) twonstitut-e ATI’-c~epentlt.tit proton pumping in vf+cles frotn which the twlogenous mt~mhatlf ATPaw had previously heen stripped. Kwonstitution was achieved with the adt-lition of the E sut)uttit.

(‘rystallization

97.4 66.2 45 31 21.5 14.4

Figure 1. SI)S/polvacrylamide gel rlectrophoreticanalysis of t,hr expression and purific~ation of the glutathionr S-transferase/E subunit fusion protein and isolation of t)hr e subunit (AIC:. MS) was carried out on thr apparatus rlertrophoresis l’hastsystem automated (I’harmac~ia LK R Biotechnology. Cppsala. Sweden). buffer \vCrt’ boiled in drnaturation Samples (40 mM-Tris. H(‘1 (pH 6.8). 10°~0 (w/v) glywrol. l+jO, (\v/v) sodium tlotlec~~lsrrl~)hat~, 400 (V/I-) /?-mrrraptorthsnol) for 5 tnin prior to rlwtrophoresis on an X”,, to 23”,, polyaqvlamidr gradient gel, (a) Total cell protein of AS2994: (b) total wll protein of AN2991 aftrr induction with 0.1 m~-isoE)roI)Sl-8-o-thiogala~~tosidt: (c) cytoplasmica fraction from an induced 10 I culturr of AX2994: (d) fusion protein purified by affinity czhromatographr on a IO ml c~olurnn of glutat,hionr-agarose and cwmprtltivr elution with IO mu-reduced glutathione: (e) fusion protein after treatment with human thrombin (Sigma (‘hrmicaal (‘0.) at 60 units/ml for 72 h at 4°C’; (f) partially-purified E subunit after rc~moval of glutathione K-transferasr and uncwt fusion prot,ein on a secwnd glutathiom-agarosr atinity column: (g) purified E subunit from size-exc4usion HPTA’: (II) molt~wlar wright markers. with weights as intlic3trtl ( X IO- 3).

Mutational analysis of E subunit function is somewhat limited since the only missense mutation reported. that affects E function, resulted in the replacement of ply:inr 48 in the mature E subunit by aspartatr ((lox (If nl.. 1987). The mutation affected assembly of the F,,F,-ATPase but this defect could be overcome by increasing the gene dosage. The however. lacked assembled enzyme. fully ,4TT’-depentlent fwot)on pumping activity hut was c~apable of carrying out osidative phosphorylation. albeit at a redu(aed rate. These effects could be ovrrcomv by t)he suhstit,ution of proline 47 with either serirre or threonine. The results were interpretrcl in terms of a change in the type of a putative p-turn. Kuki et CL/. (1988) const’ructed a series of plasmid-rnc~otlt~d (‘-terminally truncated F subunits. They detlu~d that, the region between residues 73 and X0 is important for the binding of F, to F,, and that the region between residues 80 and 93 rnediated the inhibitory action of the subunit on soluble F,. It’ would appear that residues 93 t)o 13X are not rfquired for func+ion. The gross strucsturr of the ECFoF,ATT’asr has been studied in (‘apaldi’s laboratory using the tech-

Sotra

307

nique of cry-electron microscopy. The F,-ATPase was described as six elongated protein densit.ies about 90 x x 30 A (1 Lh = 0.1 nm). forming a hexagonal barrel, the central cavity of which is ijartially occluded by an asymmetric compact protein density (Gogol et al., 1989). Monoclonal antibodies were used to demonstrate that t)he hexagorml structure consisted of alternating z and /i subunits, the relative positions of the E and 7 subunits depended on the ligands bound at the catalyt,ica sites (Cog01 et al..

1990).

(‘rystals of the F, portion of rat liver mitJochondrial ATT’ synthase have been grown (Arnzel & Peclersen. 197X), and the structure. originally present)ed at 9 A resolution (Amzel rf fd.. 1982), showed a complex having a S-fold axis of symmet,ry. inconsistent with the proposed suhunit stoichiornetry of r,b,y&. The structure at 3% .A resolution has recxently been reported (Hianchet rt r/l.. 1991) and now shows a S-fold yvstallographic axis relating the 51 and /I’ subunits. while t,htJ electron density for the minor subunits is disordered and was not interpretable. The at,omic resolut’ion structures of the individual components of F,-ATPase will t,hrreforrs corn plement thta work on the entire F, portion. IVe have begun a systematic st’udy of t)he protein from E. co/i and have cloned and expressed the 0 (cytoplasmic domain). 7. 6 and E subunits, and have commenced c*rystallizat.ion expcriment,s on these individuall?; anal as complexes. The E subunit has been crystallized in a form suitable for an S-ray structure analysis. \\‘e now report the expression, purificat,ion alid c*rystallization of the E subunit of W’F, ATPasr.

The E subunit of ECF,F,ATPasr is enc~odetl by the unc(’ gene and this gene is included on the I’stI fragment from the plasmid pAK36 (Downie et 01.. 1980). This fragment was cloned into the PstI sit,e of the I1113 plasmid rnp18 and a BcrmHl site was int’rodutaed. using site-direct’ed mutagenesis. at a site equivalent to codons two and three of the WW( reading frame. As the mat’ure E subunit, has lost, the S-terminal methionine (K’alker rt (11.. I!)#). the introduction of the RamHI site result,h in the replacement of alanine 1 by glycinc and mrthionine 2 bar serine. Digestion of the mutated plasmid with Rnrn HT produced a 625 nucleotide fragment carrying the mn~(’ gene (minus the initiating methionine codon). This fragment was cloned into the NrcmHI site of the expression vector pGE:X-2T (AMRAT) (‘orp., JIelbourne. Agust,ralia) such that the caarboxyl terminus of the Srhistosoma japonicum glutat,hionrS-transferase protein was fused to t’he S tt~rtninus of the E subunit (plasmid pAX590). Expression of the fusion protrin in strain AK2994 was first trst’ed in small sctale liquid cult’ures (see Fig. 1). A protein of about 37.000 molecular weight was protlucyd on induc*tion of the tar promoter by II’TC:. The c sut)unit although of 15.000 molwular w6ght. runs at

about

12,000 on S1~S~J~ol~:tc’r?;lctmitlr

~horcsis

(PAGE)

lvtbight of about

I’~Ppratiorc

to

give

an

t~xpwtctl

38,000 for thts fusion

thp

?f’

E subunit

(.d

I(:.

prl

rlwtro-

molrc~ular protein.

iM%S)

(‘ells culture

were harvested from an induced ten 1itr.r of strain AX2994. disrupted in a French prwsurv cell and the membranes removed hg centric fugat ion. The fusion protein was found in the supernitt ant (cvtoplasmic fraction) and was purified 1)~. first binXing to a short column of glutathioneagarosts heads. The fusion protein was elutjetl 1)~ adding reduced glutathione to the eluting buffer (see Fig. I ). The purified fusion protein was cleaved by treatment with human thrombin (Sigma (‘titmical (~‘0.. St. I,ouis, V.S.LI.)~ typically at 5 to 10 NTH I-nit,s/milligram of fusion protein for up to IO0 hours at 4°C’. Free glutathione transferase and any remaining urirut fusion protein were removed 1)y t,reatmcbnt with glutathionr-agaros(~ beads. The unbound material (containing 6 subunit) was c~onc~entrated and furt,her purified by passage through a TSK-1% high-pressure liquid chromatography (HPW) csolumn (see Fig. I ). The yield of per litre purified c subunit was about five milligrams of ~11 culture. The identity of t’he purified c subunit (,.l ZG. .V%S) was confirmed by N-terminal protein srquencGg. The rchcomhinant I: subunit, was shown to c,o-elrctrophorese with a purified sample of wild type c subunit in SI)S/PAGF. The ultra.violetj al)sorption spectrum was essent)ially t’hat published by St)ernweis ei Smith (1980) with an absorbance at 277 nm and a shoulder at about maximum 285 nm. The molar cxtinc%ion c*oeficient at) t ht absorbance maximum was 6500 Mm ’ vm ’ in water.

Punctional

assessment

qf the

E subunit

(AlG,

MBS)

The E subunit, produced by the thromhin-cleavage of the purified (XT-fusion protein has an altered ?I;-t,erminal sequence, such that alanine 1 and methionine 2 are replaced by glycine and serine respectively. In order to test whet’her t,hese substitutions affect, the function of ATP synthase that has incor-

porated the. mutjant c. t h(l I’stl t’ra~gment l.arrvinp the mutated ~L?LC(:gene (SW abo\-cx) was clont~l ‘irrt,, the unique I’stl site of thr vector pJ- membrants pwparat normal r\‘ADH and ATP-tlependrnt atrbrin fluortxswnce

qwnf*hing

wtivity.

‘I’htw

tlat.a

propwties

growth characteristics of strain,s uxprrssing (A IQ:, M2S) and wild-type E subunit and

E

AN307ti(pAX647) ‘w2413(pAN207) AN3088(p13K328)t (:rowth

of strains,

pwpmtitrn

of’ the mrmbrane

f’rwtion.

atehrin

fluor~scencr

quenching

and thr

growth of strains on surcinate minimal medium were carried out HS dewribrd hg (+ihson rt al. (1977) (:rowth yields were measured as turbidities of cwltures, using a KlettMummerson volorimetrr, after growth had ceased in mineral salts minimal medium conteining limiting (5 mM) glucose (Klrtt 100 ‘c 024 mg dry nt/ml). t Nqativr

control

stzain.

i hat

(?ystals were grown t)y vapour diffusion using t’hrb hanging-drop technique (McPherson. 19X2). Broad screening was carried out by the inc+omplrtr factorial method (Cart’er ot (:artrr, 1979). The rangt of conditions for the initial trial was (2hewn 1q selectively modifying t)hr list given by .Jancarik & Kim (1991). The variables which made up thr incomplete factorial conditions included: pH (from 4.6 to 8.5): hufl’ers (acetate, citrat,e. cacodylat’r, Hepes and Tris); precipitants (RIPI), PM: 400; PEti phosphates. 1000. PEG 6000, sotlium-J,ot,assiuln ammonium sulptiate and sodiurri-potassiu111 tartrate); added salts (cal(siurn cahloride, magnt+1m chloride and lithium sulphat,e). All wystallization trials were carried out, at 4°C;. The initial droll comprised four milligrams of protein/milli1it~re. esti mated by t,he method of Lowry et al. (19.51). in Ore hufl’er (pH 7.5), of 50 rnhl- Hepes presence 100 rnM-ammonium sulphatjc and 1 >I-phosphatca (mixed K,HPO, and NaH,PO, .2H,O). The rewrbuffer (pH 75). 100 mM-Hepes voir was

Table 1 Membrane

irrtlivatf~

the t: subunit carrying t h(l amino ;rc.itl sirtjst itutiorls AlG and h12S is func3ionally indistin~llislii~tll~, from the wild-t.J-pcb 1: subunit

Crystallization

200 mu-ammonium sulphate and 2 M-phosphate. Crystals up to 0.3 mni X 0.09 mm x @03 mm. shaped like bi-convex cylindrical lenses, grew sometime between 9 and 12 months at 4°C. X-ray diffraction photographs were taken on a precession camera, using CuK, radiation from a rotating anode generator at 50 k\: and 80 mA. The crystals, which were kept at 4°C by a cooled nitrogen gas stream. init’ially diffracted to a resolution of 2:9 8. Data beyond 6 A resolution were lost after 24 hours of X-ray exposure. The crystals are hexagonal and systematic absences and symmetry on precession photographs identify the space group as either f’6,22 or P6,22. The unit cell parameters are a = 94.9 ,A and c = 57.1 8. With a monomer in the asymmetric unit, IL the solvent content characterized b,v the ratio of the volume of the unit cell to the tnolecular weight of the protein, is 2.4 A3/Da which is near t,he average found in a survey of protein structurrs (Matt,hews, 1968). Two molecules in the asymmetric unit would place V, well outside the range observed by Matt,hews. We are in the process of’ optimizing screening for

hPiiVV

1he crystnllization condit’ions atom derivatives.

and

\Vr thank ft. Harris. (:. Hrafy and R. Taylor for skilled technical assistanc,r. and the following for generous financial support: Sir .Johrr Proud. the R,avmond E. I’urvrs Foundation. the .Jamrs XL’.Kirby Foundation. I)r C. H. 12’arrnan and thta t3rrrc.r and .Joy Reid Foundation.

References :Zmzel. I,. M. & l’edersen. P. I,. (1978). Adenosine triphosphatasr from rat liver mitochondria. .J. Rio/. C’hewL. 253, 206772069 .\mzel. 1,. 11.. McKinrq, $1.. Xaravanan. I’. B I’rdersm. of the mitochondrial F’. I,. (1982). Stru&ure F, ATI’ase at 9-A resolution. Proc. Sat. Acad. Sri., l:.s.if 79. 5x52 ~4x56. flianchrt. M.. I’srrn. Y.. Hulliben. ,J., I’edersen. 1’. I,. & Amzrl. I,. M (1991). Mitochondriaf ATP synthase: yuarternarg structure of the F, moiety at 3% A determined by X-ray diffraction anafGs. .J. /Sol. (‘hem. 266. %I 1!)7-21201. (‘arty, (‘. \V.. Jr C%(‘a&r. (‘. W. (1979). Protein crystallization using incomplete fact,orial experiments. .J. Viol. (Ihrm. 254. 12219-12223. (‘ox. (:. I%., Hatch. I,.. Webb, I)., Fimmef, A. I,.. Lin, Z.-H.. Senior. A. E. & Gibson, F. (1987). Amino acid substitutions in the E-subunit of the F,F,-XTPase of Eschrrichia I95 204.

coli.

Hiochim,

Biophys.

Acta,

890.

309

:Votes

Downie. ,J. A.. Langman, L., (:0x. (:. 13.. Yanofsky, C. & Gibson. F. (1980). Subunits of the adrnosine triphosphatase complex translated iT/ Gtro from the Escherichia coli uric operon. J. Bactrriol. 143. S-17. Dunn, S. D. (1982). The isolated y-subunit of ICscherichia coli F, ATPase binds t,he E subunit. ./. IGo/. (‘hc m. 257. 735447359. Dunn. S. I).. Zadoroznp, V. I>., Tozrr. lt. (:. & Orr. L. E:. (1987). E Subunit of Escherichia coli F,-ATI’asr: rffec%s on affinity for Aurovertin and inhibition of .\Tl’ h~drolj%. product release in unisite Biochemistry.

26, 4488-4493.

Fiflingame.

R. H. (1990). In Th,u Hnctrria: II Ireatisr on nncl function (Krulivich. T. ;\.. rtl.). 111). strllcture 345-391. .~cadrmic Press Inc.. SPS- York. (Gibson. I:.. (“ox. C:. H.. f)ownie. .J. ~1. & Ratlik. .J. (1977). .I mutation affecting a second component of the F, f)ortion of the magnesium ion-stimulated adrnosine triphosphatasr of Eschrrichia co/i Ii I:! Hiochpm. .J. 164. 193%198. Gibson, F.. Downie. ,J. A.. (‘ox. G. H. & Radik. !J. (1978). Mu-induced polarity in the WLCoperon of Eschrrichia coli. J. Bacterial. 134. 72%736. Gogol. E. l’.. Liickrn. V., Hark. T. N: (‘apaldi. R. A. (1989). Molec~ular archit,ecture of Escherichio coli F, Adenosinrtriphosl)hatase. Riochmi.str~y. 28. 470% -I-716. Gogol. E. P.. ,Johnston, E.. Aggefer. ft. & ( ‘al)aldi. R. A. (1990). Ligand-dependent, structural variations in Jkhrrichia co/i F, ATl’ase revealed b>, cr\.oelrrtron tllicroscopy. Proc. Sat. .4cnd. ,qc.i.. l’.,Y..4. 87. !cx-9589.

,Janc-a,rik. *J. & Kim. S.-H. (1991). Sparse matrix sampling: a screening method for cr@alfization of prnt,eins. .I. Appl. ~‘rystallogr. 24. 409-411. Kuki. M.. R’oumi. T.. Maeda. 11.. Amemura. .\. & Futai. Jl. (198X). Funct,ional domains of the E subunit of Eacherichia co/i H+-ATPase (F,F,). ,I Hiol. (‘hem. 263. 17437-17442. LOV ry. 0. H.. Rosebrough, IV. J., Farr. A. L. B Randall. Im. 19. 7 41. Sternweis. I’. (‘. & Smith. .J. B. (1980). (‘harac:t,erization of the inhibitory (E) subunit of the l)roton-tratnslo(~atirlg adrnosinr triphosphatasr from I~schrric~hin co/i. Iliochrnaistry. 19. FM- 531. Walktar, *J. E., Sarastr. IV. H: (:a~. X, ?J. (1!)84). The UMC operon: nuc~lrotidr seyuencr. regulation and structure of AT]‘-synthase. Biorhim. Hiophys .4cfn. 768. I64 -200.

The expression, purification and crystallization of the epsilon subunit of the F1 portion of the ATPase of Escherichia coli.

The epsilon subunit of the F0F1-ATPase from Escherichia coli has been expressed in E. coli as a fusion protein with glutathione S-transferase from the...
847KB Sizes 0 Downloads 0 Views