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A Mutant at Position 87 of the GroEL Chaperonin Is Affected in Protein Binding and ATP Hydrolysis

A Mutant at Position 87 of the GroEL Chaperonin Is Affected in Protein Binding and ATP Hydrolysis THE JOURNAL OF BIOLOGICALCHEMISTRY Vol. 270, No. 23, Issue of June 9, pp. 13956-13960, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Mutant at Position 87 of the GroEL Chaperonin Is Affected in Protein Binding and ATP Hydrolysis* (Received for publication, August 4, 1994, and in revised form, April 7, 1995) Celeste Weiss and Pierre Goloubinoff:l: From the Department of Botany, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel The highly conserved aspartic acid residue at position drolysis of ATP and, under stringent conditions, the presence of 87 of the Escherichia coli chaperonin GroEL was mu- the co-ehaperonin GroES (Goloubinoff et al., 1989; Schmidt et tated to glutamic acid. When expressed in an E. coli al., 1994a). The binding of ATP to GroEL and its subsequent 1 4 groEL mutant strain deficient for phage morphogenesis, hydrolysis occur with a high degree of cooperativity (Gray and plasmid-encoded GroEL mutant D87E restored T phage Fersht, 1991) and drive a conformational change in the chap- morphogenesis. It did not, however, reactivate the tran- eronin molecule (Saibil et al., 1993) that results in the release scription of a recombinant luciferase operon from of the folding protein (Martin et al., 1991). Mutant analysis, in Vibrio fischeri. In vitro, GroEL mutant D87E was found conjunction with x-ray structure analysis of GroEL (Fenton et to be impaired in the ability to bind nonnative proteins al., 1994; Braig et al., 1994), has provided a basis for under- and to hydrolyze ATP, resulting in less efficient refold- standing the role of individual residues in specific chaperonin ing of urea-denatured ribulose-l,5-bisphosphate carbox- functions such as ATP hydrolysis, binding of nonnative pro- ylase/oxygenase. Mutant oligomer D87E GroEL was teins, and binding of GroES. Thus, it was inferred from two able to bind GroES as efficiently as wild-type GroEL14' severe charge mutations that the aspartic acid residue at po- The conserved aspartic acid residue at position 87 lo- sition 87 of GroEL is located in the ATP-binding pocket. Sim- cated in the equatorial domain of GroEL (Braig, K., ilarly, a cluster of GroES-binding and protein-binding double Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, mutants was identified in the apical domain of GroEL, facing A., Horwich, A. L., and Sigler, P. B. (1994) Nature 371, the central cavity of the GroEL cylinder. This area was there- 578-586) is thus inferred to have a dual effect on the 14 fore inferred to constitute a part of the protein-binding site of binding of nonnative proteins to the GroEL core chap- the chaperonin (Fenton et al., 1994). eronin and on ATP hydrolysis. In this article, we mutated the highly conserved aspartic acid residue at position 87 of E. cali GroEL (Fig. 1) to glutamic acid. The GroEL protein of Escherichia coli is a member of the We found that this mutation primarily affects the binding of sequence-related family of molecular chaperones termed chap- nonnative proteins to GroEL . It also affects ATP binding and eronins, which are found in bacteria, chloroplasts, and mito- hydrolysis, but not the binding of GroES . chondria, and is related to the TCP1 family in the cytosol of MATERIALS AND METHODS eukaryotes (see Hemmingsen et al. (1988) and Lewis et al. (1992); for a review, see Hendrick and Hartl (1993)). In the cell, Plasmid pTrcESL-The groEL gene was amplified by the polymer- ase chain reaction from plasmid pKT200 (Bloomet al., 1986) using DNA GroEL and the co-chaperonin GroES facilitate the folding of oligomers 5'-TGGTCGACAAAGACGTAAAATTCGGTA-3' and 5'- proteins during synthesis and transport across membranes CCAAGCTTCTCGAGCTGGACGCACTCGC-3' that included the (Bochkareva et al., 1988; Kusukawa et al., 1989; Cheng et al., unique restriction sites Sall and HindIlI at the 3'- and 5' -ends of the 1989; Frydman et al., 1994) and are involved in the protection groEL gene, respectively. GroES was amplified from plasmid pKT200 of proteins from heat shock (Kusukawa and Yura, 1988; Martin using the DNA oligomers 5'-GACCATGGAATTCCGTCCATTGCAT- et al., 1992; Horwich et al., 1993). In vitro, GroEL and GroES GATCGC-3' and 5'-GCGTCGACCATTATCTTTATTCCTTA-3'. The amplified DNA fragments containing groES and groEL genes were facilitate the folding of a wide array of proteins (Goloubinoff et introduced into the multiple cloning site of pTrc99A (Pharmacia Bio- al., 1989; Mendoza et al., 1991; Viitanen et al., 1992). The tech Inc.) and called pTrcESL. The double mutant that resulted from GroEL oligomer is composed of two stacked rings of seven the introduction of a Sall restriction site at the N-terminal end of identical 57.3-kDa subunits with a cylindrical shape (Hendrix, GroEL (thus introducing the A to V and A to D mutations at positions 1979; Hohn et al., 1979). GroES is a heptameric ring of lO-kDa 2 and 3 of GroEL, respectively) did not exhibit different in vivo behavior subunits (Tilly et al., 1981; Chandrasekhar et al., 1986) that from the wild-type GroEL protein (Hemmingsen et al. 1988). Mutagenesis-Site-directed mutagenesis was carried out using the can bind one end (Saibilet al., 1991; Langeret al., 1992) or both polymerase chain reaction (Higuchi, 1990) with pTrcESL as a DNA ends (Azern et al., 1994b; Schmidt et al., 1994b; Llorca et al., template and the following two DNA oligomers: 5' -GCTGCAGGC- 1994) of the GroEL cylinder. 1 4 GAGGGTACCACC-3' and 5'-GGTGGTACCCTCGCCTGCAGC-3'. The The GroEL oligomer can recognize and spontaneously bind 1 4 resulting SalI-ClaI fragment, containing the D87E mutation in GroEL, nonnative proteins (Goloubinoff et al., 1989). The release of was introduced into pTrcESL, and the resulting plasmid was called bound proteins in a folding-competent state requires the hy- pTrc87. DNA sequencing of the entire groEL gene confirmed the D87E mutation. Expression and Purification of GroEL and GroES-Upon induction * This work was supported in part by Grant 00015/1 from the United of the trc promoter of pTrc87 with isopropyl-l-thio-i3-D-galactopyrano- States-Israel Binational Science Foundation, Grant 1180 from the Joint side (40 ,.,.g!ml). mutant D87E GroEL was overexpressed to a level at German-Israeli Research Program (to P. G.), and Grant 512 from the least 50-fold higher than that of background GroEL levels of the E. coli Levi Eshkol Fund (to C. W.). The costs of publication of this article were host. Purification of GroEL was as described by Azem et at. (1994a) 1 4 defrayed in part by the payment of page charges. This article must and that of GroES as described by Todd et al. (1993). therefore be hereby marked "advertisement" in accordance with 18 7 Electron Microscopy-Samples were applied to a glow-discharged, U.S.C. Section 1734 solely to indicate this fact. carbon-coated. collodion-covered 300-mesh copper grid and negatively :j:To whom correspondence should be addressed. Tel.: 972-26-585391; Fax: 972-26-584425; E-mail: [email protected]. stained with 1% aqueous uranyl acetate. Specimens were viewed with This is an Open Access article under the CC BY license. 13957 GroEL Mutant in Prot ein Binding and ATP Hydroly sis 70 S O 90 100 11 0 I I E I I I 1 ·- ,I E. c ol i AQMVKEVASKANDAAGDGTTTATVLAQAIITEGLKAVAAG VELLRSA ARTSEI CSCLI GRSLVNGLARNETS KF I ID KAPA T SDSI TE PAYRQS HELII K Wild Ty pe G roELI 4 Mu tant D87E GroEL I4 A QQ TN D K V DEVIVL NLM SR S AR I T V HGMLSH I S F IG . 2 . E lec t ro n mi c r o gra ph s of mu t an t D8 7E a n d w il d -type R N N K D Y FAK M K GroE L •.\. E K FQ C V S A F visu ally t est ed in th e dark for e m iss ion of light aft e r a 20 -h in cub a t ion L V at 30 °C . F IG. 1. Evolutionary variability a mong c ha peroni ns. Th e a m ino RESULTS a cid se q ue nce of GroEL fr om E. coli wa s a ligned in r egion 70 -110 wi th Stru cture a n d Stability of Mutant D87E-To a n a lyze th e 4 2 G ro E L, cp n6 0, a nd h sp60 s eq u e nces from th e Swi s sP rot Dat a Ba s e (199 3 ) u sing th e Pil eup a lgor it h m (ga p pen a lt y of 3) in th e 1991 Ge ne t- e ffect of t h e mutation on t h e s t r u ct ure a n d s ta b ili ty of th e ics Com p u te r G ro u p soft wa re pack a ge (De ve r e u x et aI. , 198 4 ). Bel ow cha pe r on in oligom er, purifi ed D87E GroEL " molecu les were th e E. coli se q ue nce a re lis t ed the alternative a m ino a cids fou nd a t ea ch observed by neg ative stain ele ct r on mi cr oscopy a nd compared position in a t lea s t on e of th e a ligne d se q ue nces . Und erlined resid ues with wild-typ e GroEL (Fig . 2 ). Mutant D87E oligomers are a re a ls o conse r ve d in cha pe r onins TCPl a n d TF 5 5 fro m a rchaeba cteria l4 see n to be organi zed into characteri stic t etrad ecameric doub le- (T re n t et al ., 1991 ). lay ered cylinders <Hendrix, 1979 ; Hohn et al. , 1979 ), which a re indistinguish abl e from wild-typ e GroEL . D87E di spl ayed th e a P h ilips CM 12 e le ct r on m icr oscop e ope rat i ng at 100 kV . M icr ogr aph s I4 we r e r ecord ed on Kodak S O- 163 e m u ls ion a t a n omin a l m a gnifi cation of s am e mobility as wild-t yp e GroEL on nond en aturin g 7% poly- l4 X75 ,0 00. acrylamid e ge ls and wa s a s sta ble as wild-typ e GroEL ., in th e ATPa se A cti vity-Th e hydrolysi s of ATP by Gr oEL wa s m ea sured pr es enc e of 1 1\1 urea (d a t a not s ho wn ). How ever, a t va r ia n ce as d escribed by Azem et al. (19 94 a ). Gr oEL ,., (3 IJ.M p r ot omer ) was with the wild typ e , mutant GroEL wa s h a lf di ssociated into l4 incu ba te d in 50 mxt tri eth anolam in e, pH 7.5, 10 mxt KC I, 10 mxt MgAc 2, monom ers in th e pr esenc e of 2.25 1\1 u r ea (da ta not sh own ), a n d vari ou s con centration s of [y _ :J2P]ATP (0 .0 1-5 mxt with a s pe cific sug ges t ing that under ext re me condition s , th e mutan t is mor e a ct ivity of 3 IJ.Cilmmo\) for 5 mi n at 3 7 °C. U n hy d ro lyze d ATP wa s se pa ra te d from th e "2P -la be led in or gani c pho sp hate by a dso r pt ion on prone to di ss ociation than th e wild -typ e oligomer. Fin all y , 5% ac t iva te d cha r coa l in 20 mM H"PO ,. a ccor di ng t o Ba is (197 5 ). Th e cro ss-linking with glutarald eh yd e a s describ ed by Azem et al . data for t h e velocity of th e ATPa se r eaction wer e fitt ed to th e Hill (1994 a ) confirmed that with in th e t im e sca le of th e ex pe ri me n ts eq ua t ion u sing the nonlin ea r r e gr essi on meth od of th e En zfitter com - described , > 95% of th e muta nt chaperonin wa s in th e tetra- puteri zed program a ccor d ing to Leath erbarrow ( 198 7 ). Th e following decam eric form (d ata not sh own ). Thus , th e ge ne ra l st ru ct u re cons ta n ts wer e derived (see T ab le I ): Ken' = Vma/ IGroE L J, K' = IS]".a" , of th e ch ap eronin wa s not affect ed by th e mutati on . a n d n Il = Hill coe fficie nt. Chemic al Cross -lin k ing of Chaperonin Olig omers-Th e bind in g of Geneti c Complem enta ti on wit h Wild-typ e and Mutant Gr oES to wild-typ e a n d mutant Gr oEL ,.• in solu t ion wa s m ea sured GroEL- In E . coli , expression of pla sm id -en coded (pCh Vl) lu - u sin g ch em ical cr oss -li n ki ng a t 3 7 °C wit h glutara lde hy de (0 .2 2%) and ciferase operon from V. [ischeri is u n der th e t r a ns cr ipt ion a l S DS -polya cry la m ide g e l e le ct r oph ores is as d escrib ed by Aze m et al . control of GroE ch ap eronin s (Ada r et al . , 1992 ). Thu s , wild -typ e (1994 a , 199 4b ). E . coli cell s harborin g pla smid pChVI emit 1Q4- 5_f old mor e Refolding of Rubisco'<In vitro ch a pe ro n in-d e pe n d e n t r efoldin g of visib le light in th e ea r ly stat iona ry phase than T Gr oEL Rubi sco from Rhodospirillum rubrum wa s assaye d at 25 °C as d e- S50 sc r ibe d by Gol oubinoff et al . (19 89 ). Rubi sco (20 IJ.M ) wa s den a tured in 4 mutant cells. Wh en E . coli T mutant cells h a r bor in g th e two S50 Murea a nd 10 rnxtdith ioth r eitol a n d th en di luted SO-fold into a so lu t ion comp atib le plasm ids pChVI a n d pTrc99A were s pot te d with T of 5 0 mxr T ri s , pH 7.5 , 20 mxt MgA c , 20 rnxr KCl , 1 m M dithiothreitol , bacteriophages , a plaque did not dev elop (F ig. 3A, cell la wn 2 ) 20 mxt g lu cose , 1 mxt ATP, 6 IJ.M Gr oES (p r ot orne r ), an d 3 IJ.M wild- t yp e and th e cell la wn emit te d very low level s of light (F ig. 3B , cell or mutant Gr oEL. Hexok in a se (S igm a) wa s a d de d t o a fin a l conc entra - lawn 2) . When , how ever, th e GroE-l ess pTrc99A pla smid wa s t ion of 40 u g/ rnl to s to p th e r efo lding r ea cti on a t th e ind ica te d tim e rep laced by pTrcESL, th e T cell lawn emitte d high level s of points . Rubisco a ct ivity wa s det ermin ed as d escribed by Goloubin off et S50 al. (1989). light (F ig. 3B , cell la wn 1) and dev eloped a plaqu e (F ig. 3A , cell In hibition of Protein Refoldin g-Pi g h eart mitochondri a l m a late d e- lawn 1). Wh en pTrcESL was r ep lac ed by pTrc87 containing hydrogena se (9 IJ.M m on om er; Boehringer Mannh eirn ), d en atured in 4.5 GroEL with t h e D87E muta tion , a n interm ediat e ph en otyp e ~I urea for 3 h a t 25 °C , wa s di luted 70 -fold into a r efoldin g so lu t ion was observed: th e cell la wn emit te d only low lev els oflight (F ig. contai ning in cr ea sing a m oun ts of mutant or wild- t yp e GroEL , 10 mxt 3B , cell la wn 3 ) but s uccess fu lly develop ed a T plaq u e (F ig. 3A , KCl, 20 mxi MgAc 5 0 mM tri eth an olam in e , pH 7.5, a n d 2 ms: dithio - 4 2, cell lawn 3) . Thus , th e m utant ch ap eronin is partia lly fun c- t hreitol. Ma late dehydrogenase a ct ivity wa s assayed at 25 °C in 150 mn K + pho sphate buffe r , pH 7.5, 0.5 mx: oxa lace ta te, 2 m M dithiothre itol , tional in viv o. and 0.2 mg / m l NADH by m ea surin g th e time-d e pendent ch a nge in t h e Ch ape ronin-ass isted Refolding of Rubisco in Vitro -When ab sorption of mon ochrom atic light a t 340 nm (Mill e r et aI. , 1993 ). com pa re d with wild -typ e GroEL , the chaperonin -dep end ent I4 Geneti c Complem entation of Phage Morph ogenesi s-The E. coli mu- r efolding of t h e Rubi sco e nzy me by mutant D87E ex h ibite d a tan t s t rai n TH aDca r ries a chrom oso mal mutation in th e groE L ge ne that slower r ate and r eached a maximum r ecovery of - 25% th at pr events maturation of A-bacterioph a ge prohead s (Taka no a nd obt ain ed with the wild-typ e chaperonin (F ig. 4 ). Kakefuda , 1972 ). T " wa s tran sform ed with e it he r pTrcESL or pTrcS7 , H a a nd p h a ge morph ogenes is wa s t ested u sing a "s pot test" for plaque I n h ibit ion ofSpontan eou s Prot ein Refolding-At 25 cC, urea- formation with T bacterioph ag e a ccor d ing to Reve l ( 19 80 ) a n d Gr een er denatured ma late dehyd r ogen a se can r efold s pon t a ne ous ly into et al . (199 3). native enzyme (Mill er et al. , 1993 ). In th e pr es enc e of GroEL , I4 Geneti c Complem entation of Lu ciferase Tran script ion-In E. coli , th e however, th e malate dehydrogenase folding interm ediates bind tran scription of a pl a smid-encod ed lumin escenc e (l ux ) operon from to th e chaperonin and are pr evented from r efold ing into an Vibri o [ischeri , pCh VI (Ul it zu r a nd Kuhn , 198 8 ), requi r es hi gh lev e ls of active enzy m e . Fig . 5 sh ows that th e concen tration of D87E Gr oEL a nd GroES (Ad a r et al. , 1992 ). Wi ld -t yp e E. coli cell s carryin g 4 5 pChV l e m it le ve ls of visibl e light 10 - times high er th an T H aD ce ll s GroEL required to inhibit h a lf the s pon t a ne ou s r efolding of l4 ca r ry ing pChVl (Ad a r et aI. , 1992 ). Ce ll la w n s of T carryi n g t h e eao m alate dehydrogenase is four tim es hi gher t han for wild-t ype comp atib le pl a smid pTrcESL or pTrc87 in a d d iti on t o pChV l wer e GroEL J4" Th is indic ates th at t h e spontaneo us pa ssi ve binding of nonnativ e protein is deficient in th e mutant. Stabi lit y of th e Binary Com p lex- Wh en wild -typ e GroEL l4 , T h e a bbrevia t ion u s e d is : Rubisco , r ibul ose -l ,5-bi sphosphate car- boxyl a s e/oxy genase . wa s fir st ch all enged with nonnative Rubi sco, incubated with 13958 GroEL Mutant in Protein Binding and ATP Hydroly sis A B ~ 1 0 0 J: ::E Cl c: 'C 6 0 Gl a: !II :::s Ql c: 11l FIG. 3. Genetic c omp lementation of a GroEL m utant h o s t 2 0 strai n. Th e E . coli Gr oEL mutant s t ra in T (Ta ka no a n d Ka ke fuda , 8 5 0 0 1972) was trans formed with a pla sm id , pGhV1 , con ta i ni ng th e lu cifer - CIl ase ope r on fr om V. fi scheri (U lit zu r a n d Kuhn , 1988 ) a n d with a com - patibl e pla sm id : wil d-ty pe pTrcESL (cell law n 1), G r oE -less control pl a smid pT rc99A (cell la wn 2), or mutant pT rc87 (cell la wn 3 ). Ba ct e ri al 4 5 0 2 3 lawns on LB a ga r plates we re s potte d wit h 3 p.1of T ', bacteriophage (l0" [Gr oE L]14/MDH Rati o plaque-forming u n it s/ml ), Aft er a 20-h in cu ba t ion a t 30 °G, th e bacteri al lawn wa s visuali zed for th e formation of a ba ct eri oph a ge pl aqu e in th e F IG. 5 . Inhibition o f s p onta neo us m alat e de hydroge nase re - li ght (A) and in t h e dark (B ). folding b y w i l d- type a n d mu t a n t D 8 7E GroE L. Ma late deh yd r o- ge nase (MDH; 9 P.M ) in 4 .5 M urea wa s diluted into a r e foldi ng solu t ion conta ini ng increasing concentration s of wild-typ e (e) or mu t ant D8 7E (0) G r oEL .... Ma late deh ydrogen a se a ct ivi ty wa s measured a s describ ed ::e e..... und er "Ma t e r ia ls a nd Method s" a fte r 135 min of r efolding a t 25 °G. e- Gl Gl a: >- ... Ql ~ 80 .!!! ..c o :::s Ql a: a: Gl o 60 !II III ..c Gl a: ~ 40 Ql 0 5 1 0 1 5 2 0 25 80 § 2 0 Ql Time (m in) a: FIG. 4 . Time cou rse o f Rubis c o r efolding b y wild-t ype a n d mu- t ant D87E G roE L. Rubi s co (20 p.M ) , den atured in 4 Murea , wa s dil u t ed 80 -fold into a refoldin g m ixture conta ining 6 p.M GroE S , 1 mMATP , a n d 20 30 40 50 60 0 10 3 p.M wi ld -typ e ( e ) or mutant D87E (0) Gro E L as describ ed under Tim e ( m i n) "Ma te rials a n d Method s ." Aliquots wer e r emov ed a t variou s t ime inter- va ls , and Rubisco a ct ivi ty was det ermi ned as desc rib ed by Goloub inolT F IG . 6. Rubis c o recovery after incubation wi t h or without et al . ( 198 9). T h e perc ent r ecovery is ca lculated r elative to th e a ct iv ity AT P . Ur ea -den atured Rubi sco wa s di luted as descr ibed in th e lege n d of of th e sa me conce n t ra tio n of n ati ve Rubi sco . Fig. 4 in th e pr esenc e of wild -typ e (. a n d e ) or mutant D87E (0 a n d 0 ) GroEL .... Binary comp lex es were in cub a ted for in cr ea s in g a mo u nts of ATP for in creasing amo u nts of time, and then supplemented tim e in th e pr es en ce (0 a nd e ) or a bse nce (0 a n d . ) of ATP pri or to t he with GroES to initiate re folding, no si gni fica n t loss of recov - a ddit ion of Gro ES (6 p.M). Th e Gro ES -de pe nde nt re foldi ng reaction was a llowe d to pr ogr ess for 15 min a nd th en wa s a rreste d with glu cose a nd ere d Rubisco wa s observed (F ig. 6 , e ). In contrast, when h exokina se a s in th e legend of Fi g . 4. Th e per cent r ecove ry is cal cu lated Ru bis co-bou nd mutant D87E GroEL was incubated with ATP fro m th e Rubi sco ac tiv ity re covere d wh e n ATP a nd Gro ES we re pr o- for increasing amo unts of tim e and th en s upp lemented with vid ed a t th e tim e of t h e Rub isco diluti on . GroES , a sign ifica n t time- dep endent loss of recov erable Rubi sco wa s obs erve d (F ig. 6 , 0 ). If, h owever , the initial incu- bation was carried out in the absence of ATP , no los s of recov - typ e GroE L . Mor eover, ATP was hydr olyzed by t h e mu tant in erable Rub isco activity was observed, either for wild -ty pe or a noncooperati ve m anne r . However, GroES inhibited ATP hy- mutant D87E GroE L (Fig. 6 , • an d D , re spectively ). Th ese dr olys is sim ila r ly in mutant a n d wild-type GroEL (Ta ble 1). re su lts further indi ca t e that the bin ding an d rebinding of non - Th is su gges ts t hat mutant D87E is deficie nt in t h e ability to n a t ive p r ot ein to t he chaperonin a re a ffect ed by t he D87E bi nd a nd h ydro lyz e ATP, but not in th e ab ilit y to bind GroES • mu t a ti on . B inding of GroES to Mutant D87E and Wild -typ e ATPase A ctivity-The r ate of ATP hydr olysis by m utant GroEL I..-The binding of GroES t o mutan t D87E or wild- typ e D87E is h alf that of wild-type GroE L (Fig. 7). Ta ble I sum - GroEL oligomers in the pr esen ce of ATP was meas ured usin g 1 4 14 marizes t he kinetic parameters for t h e ATPase activity of mu- che mical cro ss -linking wit h glutaral de hyd e a nd SDS-po lyacryl- tant and wild -typ e Gro EL • Th e affinity of AT P for mu t a n t amid e gel electrophore sis a s de scrib ed by Azem et al. (l994b ). 1 4 In th e presence of a saturating concentration of ATP , GroES D87E was nea rly a n or der of m agnitu de lower t han for wild- 7 OroEL Mutan t in Protein B inding and ATP Hy d roly sis 13959 7 0 C 1 2 3 4 5 67 8 . ) ... G> 5 0 :Gb"r ~HW6 ~~ ~ i )2: -0 ... GroELl 4 a. ... G> a. Ul ... G> D1 2 3 4 5 67 8 B 1 2 3 4 5 6 7 2 0 c: ... ::J I- :Gb" r~HW 6~ ~ ~P2 : Gro ELl4 0 4 8 12 16 Time ( mi n ) FIG. 7. Time -d epe n d ent h y d r ol y si s o f ATP b y w il d -typ e (e) and mutant (0) GroEL. F IG . 8 . B indin g of GroES to w il d -t yp e a n d m uta nt GroEL14' Wild-type GroE L •• (3 J.tM pr ot omer ) (A) or D87E GroEL (B ) was TABLE I 14 inc uba t ed wit h 1 mMAT P a n d in cr ea s in g concen tr ations of Gr oES . T he Chara cteristi cs of A TPa se activity of wild-typ e r a t io bet ween GroES a n d Gro E L pr otom er s was 0, 0 .125 , 0 .25, 0.50 , and m utant D87E GroEL 0.75 ,1.0, a n d 2.0 in lanes 1- 7, r esp ect ively. Wild-type Gro EL (3 J.tM ) (C) T he K ' a nd n il va lue s for wild-type and m ut ant D87E GroEL wer e the or D87E GroEL (D ) was incuba t ed wit h a n exce ss of GroES (6 J.t ~I ) a nd a ve ra ge of eight a n d s ix experi me n t s , respec t ively. Val u es for th e inh i- InCrM siri.g con cen t r a t ion s of A'1'P (0,1 ,3 , 5, 10, 50,100, a n d 500 J.tMATP b it ion by UroES of t.he UroE L A'I'Pase wer e t h e average of four exp er - in lan es 1- 8 , re spec tive ly). Cross-lin kin g of cha peroni n h et er o-oli- im ents . Ken' va lu es are from a r ep resen t a ti ve experi me n t i n whi ch t h e gome rs with 0.22% glutara lde hy de , SDS -polyacryla mid e gel electro- pu rifi ed mu t a nt D87E pr otein wa s devoid of det ect a ble degradation ph or es is , a n d Coomassie Blu e sta ining wa s accordi ng to Azem et at. produ ct s a s j udg ed by n a ti ve SDS gels (da ta not show n ). (1994a). Cooperat ivity Inhibition by J(O GroEL K ent GroES ( n il) recovery of Ru bisco by t he mutant, in th e pr esen ce of ATP a nd min - mM % GroES?, was - 25% t hat of t he wild-ty pe chap er onin (Fig. 4). Wild-typ e 6.0 26.1 := 7.1 1.80 := 0 .14 38 .8 := 9.0 Th e bin a ry com plex bet ween mutant cha pe roni n a nd D87E 3.2 227 := 6 1 0 .88 := 0 .10 50.8 := 4.0 Rubi sco was a lso less stable t h an the wild-ty pe bin a ry complex whe n inc uba te d for inc r ea sin g peri ods of t im es wit h ATP . Ma r ti n et al. (199 1) sugges te d th a t ATP hydrolysi s , in t he h a s t he sa me a ffin ity for wild-ty pe Gro E L (Fig. SA) as for 1 4 a bse nce of Gro ES, ca n ca use bound pr oteins t o und er go mu lt i- mutant D87E Gro E L (F ig. 8B ). However , in t he p res enc e of a ple fu t ile cycles of re lease a n d rebindin g to Gr oEL . Whil e t he saturati ng conce ntration of Gro ES ?, Gro ES bin di ng r equ ir es wild-ty pe bin a ry com plex a p pea re d t o wit hs ta nd multiple 5.7-fold les s ATP to bind wild- typ e Gro E L (F ig. sc: compar ed ATP a se-dr iven cycles of protein r elea se a nd re bi ndi ng wit hout with mu t an t D87E (F ig. 8D). Th e effective concen t rations of a significa n t loss of recover abl e Rubi sco, the mu t ant binary ATP neces sa ry for th e bindin g of Gro ES? to h a lf of t he Gr oEL com plex was signi fica n tly dest abilized by ATP hyd r olysis (Fig. oligome rs wer e 34 a n d 6 /L M for mu t a n t a n d wild- t yp e Gr oEL , 5 ), suggesting th at the r ebi nding of Rubisco to t he mu t a n t is re s pectively. Th is s uggests t hat fu nct iona l bindin g of Gr oES ? t o less successfu l t h a n to th e wil d-ty pe molecul e. Thi s dem on- Gro EL is not dir ectly a ffecte d by the D87E mutation , bu t st rates t hat, alth ough t he mu t ation is in t he ATP -binding rather th ro ug h t h e decr ea s ed a ffinity of t he mutant for ATP . pocket , th e AT P hyd r olysis mech ani sm is fu nctional an d is DISCUSSI ON capable of dri vin g r ever sibl e conforma ti on al cha nges in the In thi s wor k , t he h ighl y conserve d resi due Asp- 87 of GroEL mu tant chap er onin . Furthermo re , it confirms th at mutan t D87E ch ap er onin s was mutated t o glu t a mic ac id. In a Gr oEL -defi- is primaril y a ffected in its ability to init ia lly bind a nd to subse - cie nt E . coli host cell , pla smid-enc oded mutant D87E r est or ed qu ently r ebind nonnat ive pr ot ein s duri ng AT P-drive n cycles . ph a ge morph ogen esi s as efficien tly as wil d-ty pe Gr oEL , but not A ki ne tic a na lysis of ATP depend en ce curves of t he Gro E L t he ex pressio n of the lu x opero n , indi catin g t hat th e mutant ATP a se showe d th a t t he maxim a l ATP a se ac tivity of th e mu- chape ro nin is partiall y fun cti onal in vivo. Whil e under ext re me ta n t wa s 50% th at of t he wild ty pe a nd th a t t he a ffinity of ATP conditio ns, t he D8 7E mu t an t m ay be less stable in vit ro th an for mu t ant D87E wa s a n or de r of m agn itude lower t ha n for wil d-ty pe Gro E L , it n everth eles s asse mbled in t o a functi on al wild -t yp e Gr oEL Fen t on et al. (1994) showe d t hat two differ- 14 w oligomer with t he sa me a pparent st r uc t u re as wil d-t ype ent charge muta t ion s a t t he sa me positi on , D87K a n d D87N, Gro E L . Mor eover , mu t ant ch ap er on in D87E retaine d, a lbeit r esulted in a com plete loss of ATP a se activity a nd, conse - at various levels , a ll t he cha pe ron in func ti ons , thereby a llowi ng qu ently, of t he prot ein r efoldi ng ac t ivi ty a n d Gr oES binding a n a na lys is of t he r ela t ionship bet ween ATP h ydro lysi s , Gr oES ability. Th e effects of our D87E mu t ation as well as of D87K binding, p rotei n bindin g, a n d pr otein folding in t he olig omer. a n d D87N a re comp a t ibl e wit h t he st ruc t u re a na lysis fr om Th e sponta neo us form a t ion of the cha pe ro ni n-m a la te deh y- x-ray crystallography, wh ich pla ces Asp -87 in t he ATP-binding drogen a se bin a r y complex was four t imes less efficient for pocket of Gro E L (Br a ig et al., 1994; Fen t on et al., 1994; Kim et mu t ant D87E t ha n for wil d-ty pe Gr oEL indicating that mu- al . , 1994 ). Th e fac t that a s ign ifica nt level of pr ot ein r efoldin g tant D87 E h a s a lower a ffinity for nonn ati ve pr ot ein s . Cons ist- was a ch ieve d by a mutant la cking cooper a ti vity in ATP hydr ol- ent wit h t he 4-fold re ductio n in t h e a bility of the mutant cha p- ysis suggests t hat cooperativi ty of ATP hydrolysis may not be ero ni n to bin d nonn ati ve ma la t e deh ydrogen a se , the m axim al an a bso lute r equi r em ent of t he chape roni n -ass isted pr otein 13960 GroEL Mutant in Protein Binding and ATP Hydrolysis L., and Sigler, P. B. (1994) Nature 371, 578-586 folding mechanism, as previously suggested (Bochkareva et aZ. , Chandrasekhar, G. N., Tilly, K, Woolford, C., Hendrix, R, and Georgopoulos, C. 1992; Jackson et aZ., 1993; Langer et aZ., 1992). (1986) J. Bioi. Chem. 261, 12414-12419 Higher concentrations of ATP were required for the binding Cheng, M. Y., Hartl, F.-V., Martin, J., Pollock, R A., Kalousek, F., Neupert, W., Hallberg, E. M., Hallberg, R L., and Horwich, A. L. (1989) Nature 337, 620-625 of GroES to the mutant compared with the wild-type chapero- Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12,387-395 nin oligomer (Fig. 8, C and D). This can be explained by the Fenton, W. A., Kashi, Y., Furtak, K, and Horwich, A. L. (1994) Nature 371, 614-619 lower affinity of mutant D87E for ATP. A titration of the Frydman, J., Nimmesgern, E., Ohtsuka, K, and Hartl, F.-V. (1994) Nature 370, GroES-dependent binding of GroES to GroEL in the pres- 7 14 111-117 ence of a saturating concentration of ATP showed that GroES Goloubinoff, P., Christeller, J. T., Gatenby, A. A., and Lorimer, G. H. (1989) Nature 342, 884 - 889 has the same affinity for wild-type GroEL as for mutant Gray, T. E., and Fersht, A. R (1991) FEBS Lett. 292, 254-258 D87E GroEL (Fig. 8, A and B). This was confirmed independ- Greener, T., Govezensky, D., and Zamir, A. (1993) EMBO J. 12,889-896 ently when the same concentration of GroES inhibited half of Hemmingsen, S. M., Woolford, C., van der Vies, S. M., Tilly, K, Dennis, D. T., Georgopoulos, C. P., Hendrix, R W., and Ellis, R J. (1988) Nature 333, 330-334 the ATPase activity in the wild-type oligomer compared with Hendrick, J. P., and Hartl, F-V. (1993) Annu. Rev. Biochem. 62,349-384 the mutant oligomer (Table I). Furthermore, the binding of Hendrix, R W. (1979) J. Mol. Bioi. 129, 375-392 Higuchi, R (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, GroES to the mutant was functional since it ultimately re- M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) pp. 177-184, sulted in the refolding of Rubisco. Academic Press, Inc. San Diego, CA A cluster of GroEL mutants, all impaired in the ability to Hohn, T., Hohn, B., Engel, A., and Wurtz, M. (1979) J. Mol. BioI. 129,359-373 Horwich, A. L., Low, K B., Fenton, W. A., Hirshfield, 1. N., and Furtak, K (1993) bind nonnative proteins, was shown to be located in the apical Cell 74, 909-917 domain of GroEL, facing the upper central cavity of the oli- Jackson, G. S., Staniforth, R A., Halsall, D. J., Atkinson, T., Holbrook, J. J., gomer (Braig et aZ., 1994; Fenton et aZ., 1994). Remarkably, all Clarke, A. R, and Burston, S. G. (1993) Biochemistry 32, 2554-2563 Kim, S., Willison, K R, and Horwich, A. L. (1994) Trends Biochem. Sci. 19, the protein-binding mutants were also found to be impaired in 543-548 the ability to interact with GroES , suggesting that nonnative Kusukawa, N., and Yura, T. (1988) Genes & Deu. 2,874-882 Kusukawa, N., Yura, T., Ueguchi, C., Akiyama, Y., and Koreaki, 1. (1989) EMBO proteins and GroES compete for common sites in the inner J. 8, 3517-3521 face of the apical domain of the GroEL core oligomer (Fenton 1 4 Langer, T., Pfeifer, G., Martin, J., Baumeister, W., and Hartl, F.-V. (1992) EMBO et aZ., 1994). Our findings suggest that the protein-binding sites J. 11,4757-4765 Leatherbarrow, R J. (1987) Enzfitter Program, Elsevier Science Publishers B. V., are not necessarily located in the apical region of the central Amsterdam cavity, but may be located on the external envelope of the Lewis, V. A., Hynes, G. M., Zheng, D., Saibil, H., and Willison, K (1992) Nature GroEL cylinder, in the equatorial domain of GroEL (Braig et 358, 249-252 1 4 Llorca, 0., Marco, S., Carrascosa, J. L., and Valpuesta, J. M. (1994) FEBS Lett. al., 1994). This finding is of particular importance in view of 345, 181-186 recent observations that symmetric GroEL ) 2 oli- 14(GroES7 Martin, J., Langer, T., Boteva, R, Schramel, A., Horwich, A. L., and Hartl, F.-V. (1991) Nature 352, 36-42 gomers, in which both access ways to the central cavity are Martin, J., Horwich, A. L., and Hartl, F.-V. (1992) Science 258, 995-998 obstructed, are nevertheless fully functional chaperonins capa- Mendoza, J. A., Rogers, E., Lorimer, G. H., and Horowitz, P. M. (1991) J. Bioi. ble of successfully assisting the refolding of nonnative proteins Chem. 266, 13044-13049 Miller, A. D., Maghlaoui, K, Albanese, G., Kleinjan, D. A., and Smith, C. (1993) (Azem et aZ., 1994b). Biochem. J. 291, 139-144 Revel, H. (1980) J. Virol. 33, 366-376 Acknowledgments-We thank Martin Kessel for providing the elec- Saibil, H., Dong, Z., Wood, S., and auf der Mauer, A. (1991) Nature 353, 25-26 tron micrograph in Fig. 2, S. Ulitzur for plasmid pChVl, G. Lorimer for Saibil, H. R, Zheng, D., Roseman, A. M., Hunter, A. S., Watson, G. M. F., Chen, S., purified Rubisco, and S. Diamant and A. Azem for critical review and auf der Mauer, A., O'Hara, B. P., Wood, S. P., Mann, N. H., Barnett, L. K, and discussions. Ellis, R J. (1993) Curro Bioi. 3, 265-273 Schmidt, M., Buchner, J., Todd, M., Lorimer, G., and Viitanen, P. V. (1994a) REFERENCES J. Bioi. 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M., Flynn, G. C., Rothman, J. E., and Girshovich, A. Ulitzur, S., and Kuhn, J. (1988) J. Biolumin. Chemilumin. 2, 81-93 S. (1992) J. BioI. Chem. 267, 6796-6800 Viitanen, P. V., Gatenby, A. A., and Lorimer, G. H. (1992) Protein Sci. 7,363-369 Braig, K, Otwinowski, Z., Hegde, R, Boisvert, D. C., Joachimiak, A., Horwich, A. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

A Mutant at Position 87 of the GroEL Chaperonin Is Affected in Protein Binding and ATP Hydrolysis

Weiss, Celeste; Goloubinoff, Pierre
Journal of Biological Chemistry – Jun 1, 1995
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THE JOURNAL OF BIOLOGICALCHEMISTRY Vol. 270, No. 23, Issue of June 9, pp. 13956-13960, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Mutant at Position 87 of the GroEL Chaperonin Is Affected in Protein Binding and ATP Hydrolysis* (Received for publication, August 4, 1994, and in revised form, April 7, 1995) Celeste Weiss and Pierre Goloubinoff:l: From the Department of Botany, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel The highly conserved aspartic acid residue at position drolysis of ATP and, under stringent conditions, the presence of 87 of the Escherichia coli chaperonin GroEL was mu- the co-ehaperonin GroES (Goloubinoff et al., 1989; Schmidt et tated to glutamic acid. When expressed in an E. coli al., 1994a). The binding of ATP to GroEL and its subsequent 1 4 groEL mutant strain deficient for phage morphogenesis, hydrolysis occur with a high degree of cooperativity (Gray and plasmid-encoded GroEL mutant D87E restored T phage Fersht, 1991) and drive a conformational change in the chap- morphogenesis. It did not, however, reactivate the tran- eronin molecule (Saibil et al., 1993) that results in the release scription of a recombinant luciferase operon from of the folding protein (Martin et al., 1991). Mutant analysis, in Vibrio fischeri. In vitro, GroEL mutant D87E was found conjunction with x-ray structure analysis of GroEL (Fenton et to be impaired in the ability to bind nonnative proteins al., 1994; Braig et al., 1994), has provided a basis for under- and to hydrolyze ATP, resulting in less efficient refold- standing the role of individual residues in specific chaperonin ing of urea-denatured ribulose-l,5-bisphosphate carbox- functions such as ATP hydrolysis, binding of nonnative pro- ylase/oxygenase. Mutant oligomer D87E GroEL was teins, and binding of GroES. Thus, it was inferred from two able to bind GroES as efficiently as wild-type GroEL14' severe charge mutations that the aspartic acid residue at po- The conserved aspartic acid residue at position 87 lo- sition 87 of GroEL is located in the ATP-binding pocket. Sim- cated in the equatorial domain of GroEL (Braig, K., ilarly, a cluster of GroES-binding and protein-binding double Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, mutants was identified in the apical domain of GroEL, facing A., Horwich, A. L., and Sigler, P. B. (1994) Nature 371, the central cavity of the GroEL cylinder. This area was there- 578-586) is thus inferred to have a dual effect on the 14 fore inferred to constitute a part of the protein-binding site of binding of nonnative proteins to the GroEL core chap- the chaperonin (Fenton et al., 1994). eronin and on ATP hydrolysis. In this article, we mutated the highly conserved aspartic acid residue at position 87 of E. cali GroEL (Fig. 1) to glutamic acid. The GroEL protein of Escherichia coli is a member of the We found that this mutation primarily affects the binding of sequence-related family of molecular chaperones termed chap- nonnative proteins to GroEL . It also affects ATP binding and eronins, which are found in bacteria, chloroplasts, and mito- hydrolysis, but not the binding of GroES . chondria, and is related to the TCP1 family in the cytosol of MATERIALS AND METHODS eukaryotes (see Hemmingsen et al. (1988) and Lewis et al. (1992); for a review, see Hendrick and Hartl (1993)). In the cell, Plasmid pTrcESL-The groEL gene was amplified by the polymer- ase chain reaction from plasmid pKT200 (Bloomet al., 1986) using DNA GroEL and the co-chaperonin GroES facilitate the folding of oligomers 5'-TGGTCGACAAAGACGTAAAATTCGGTA-3' and 5'- proteins during synthesis and transport across membranes CCAAGCTTCTCGAGCTGGACGCACTCGC-3' that included the (Bochkareva et al., 1988; Kusukawa et al., 1989; Cheng et al., unique restriction sites Sall and HindIlI at the 3'- and 5' -ends of the 1989; Frydman et al., 1994) and are involved in the protection groEL gene, respectively. GroES was amplified from plasmid pKT200 of proteins from heat shock (Kusukawa and Yura, 1988; Martin using the DNA oligomers 5'-GACCATGGAATTCCGTCCATTGCAT- et al., 1992; Horwich et al., 1993). In vitro, GroEL and GroES GATCGC-3' and 5'-GCGTCGACCATTATCTTTATTCCTTA-3'. The amplified DNA fragments containing groES and groEL genes were facilitate the folding of a wide array of proteins (Goloubinoff et introduced into the multiple cloning site of pTrc99A (Pharmacia Bio- al., 1989; Mendoza et al., 1991; Viitanen et al., 1992). The tech Inc.) and called pTrcESL. The double mutant that resulted from GroEL oligomer is composed of two stacked rings of seven the introduction of a Sall restriction site at the N-terminal end of identical 57.3-kDa subunits with a cylindrical shape (Hendrix, GroEL (thus introducing the A to V and A to D mutations at positions 1979; Hohn et al., 1979). GroES is a heptameric ring of lO-kDa 2 and 3 of GroEL, respectively) did not exhibit different in vivo behavior subunits (Tilly et al., 1981; Chandrasekhar et al., 1986) that from the wild-type GroEL protein (Hemmingsen et al. 1988). Mutagenesis-Site-directed mutagenesis was carried out using the can bind one end (Saibilet al., 1991; Langeret al., 1992) or both polymerase chain reaction (Higuchi, 1990) with pTrcESL as a DNA ends (Azern et al., 1994b; Schmidt et al., 1994b; Llorca et al., template and the following two DNA oligomers: 5' -GCTGCAGGC- 1994) of the GroEL cylinder. 1 4 GAGGGTACCACC-3' and 5'-GGTGGTACCCTCGCCTGCAGC-3'. The The GroEL oligomer can recognize and spontaneously bind 1 4 resulting SalI-ClaI fragment, containing the D87E mutation in GroEL, nonnative proteins (Goloubinoff et al., 1989). The release of was introduced into pTrcESL, and the resulting plasmid was called bound proteins in a folding-competent state requires the hy- pTrc87. DNA sequencing of the entire groEL gene confirmed the D87E mutation. Expression and Purification of GroEL and GroES-Upon induction * This work was supported in part by Grant 00015/1 from the United of the trc promoter of pTrc87 with isopropyl-l-thio-i3-D-galactopyrano- States-Israel Binational Science Foundation, Grant 1180 from the Joint side (40 ,.,.g!ml). mutant D87E GroEL was overexpressed to a level at German-Israeli Research Program (to P. G.), and Grant 512 from the least 50-fold higher than that of background GroEL levels of the E. coli Levi Eshkol Fund (to C. W.). The costs of publication of this article were host. Purification of GroEL was as described by Azem et at. (1994a) 1 4 defrayed in part by the payment of page charges. This article must and that of GroES as described by Todd et al. (1993). therefore be hereby marked "advertisement" in accordance with 18 7 Electron Microscopy-Samples were applied to a glow-discharged, U.S.C. Section 1734 solely to indicate this fact. carbon-coated. collodion-covered 300-mesh copper grid and negatively :j:To whom correspondence should be addressed. Tel.: 972-26-585391; Fax: 972-26-584425; E-mail: [email protected]. stained with 1% aqueous uranyl acetate. Specimens were viewed with This is an Open Access article under the CC BY license. 13957 GroEL Mutant in Prot ein Binding and ATP Hydroly sis 70 S O 90 100 11 0 I I E I I I 1 ·- ,I E. c ol i AQMVKEVASKANDAAGDGTTTATVLAQAIITEGLKAVAAG VELLRSA ARTSEI CSCLI GRSLVNGLARNETS KF I ID KAPA T SDSI TE PAYRQS HELII K Wild Ty pe G roELI 4 Mu tant D87E GroEL I4 A QQ TN D K V DEVIVL NLM SR S AR I T V HGMLSH I S F IG . 2 . E lec t ro n mi c r o gra ph s of mu t an t D8 7E a n d w il d -type R N N K D Y FAK M K GroE L •.\. E K FQ C V S A F visu ally t est ed in th e dark for e m iss ion of light aft e r a 20 -h in cub a t ion L V at 30 °C . F IG. 1. Evolutionary variability a mong c ha peroni ns. Th e a m ino RESULTS a cid se q ue nce of GroEL fr om E. coli wa s a ligned in r egion 70 -110 wi th Stru cture a n d Stability of Mutant D87E-To a n a lyze th e 4 2 G ro E L, cp n6 0, a nd h sp60 s eq u e nces from th e Swi s sP rot Dat a Ba s e (199 3 ) u sing th e Pil eup a lgor it h m (ga p pen a lt y of 3) in th e 1991 Ge ne t- e ffect of t h e mutation on t h e s t r u ct ure a n d s ta b ili ty of th e ics Com p u te r G ro u p soft wa re pack a ge (De ve r e u x et aI. , 198 4 ). Bel ow cha pe r on in oligom er, purifi ed D87E GroEL " molecu les were th e E. coli se q ue nce a re lis t ed the alternative a m ino a cids fou nd a t ea ch observed by neg ative stain ele ct r on mi cr oscopy a nd compared position in a t lea s t on e of th e a ligne d se q ue nces . Und erlined resid ues with wild-typ e GroEL (Fig . 2 ). Mutant D87E oligomers are a re a ls o conse r ve d in cha pe r onins TCPl a n d TF 5 5 fro m a rchaeba cteria l4 see n to be organi zed into characteri stic t etrad ecameric doub le- (T re n t et al ., 1991 ). lay ered cylinders <Hendrix, 1979 ; Hohn et al. , 1979 ), which a re indistinguish abl e from wild-typ e GroEL . D87E di spl ayed th e a P h ilips CM 12 e le ct r on m icr oscop e ope rat i ng at 100 kV . M icr ogr aph s I4 we r e r ecord ed on Kodak S O- 163 e m u ls ion a t a n omin a l m a gnifi cation of s am e mobility as wild-t yp e GroEL on nond en aturin g 7% poly- l4 X75 ,0 00. acrylamid e ge ls and wa s a s sta ble as wild-typ e GroEL ., in th e ATPa se A cti vity-Th e hydrolysi s of ATP by Gr oEL wa s m ea sured pr es enc e of 1 1\1 urea (d a t a not s ho wn ). How ever, a t va r ia n ce as d escribed by Azem et al. (19 94 a ). Gr oEL ,., (3 IJ.M p r ot omer ) was with the wild typ e , mutant GroEL wa s h a lf di ssociated into l4 incu ba te d in 50 mxt tri eth anolam in e, pH 7.5, 10 mxt KC I, 10 mxt MgAc 2, monom ers in th e pr esenc e of 2.25 1\1 u r ea (da ta not sh own ), a n d vari ou s con centration s of [y _ :J2P]ATP (0 .0 1-5 mxt with a s pe cific sug ges t ing that under ext re me condition s , th e mutan t is mor e a ct ivity of 3 IJ.Cilmmo\) for 5 mi n at 3 7 °C. U n hy d ro lyze d ATP wa s se pa ra te d from th e "2P -la be led in or gani c pho sp hate by a dso r pt ion on prone to di ss ociation than th e wild -typ e oligomer. Fin all y , 5% ac t iva te d cha r coa l in 20 mM H"PO ,. a ccor di ng t o Ba is (197 5 ). Th e cro ss-linking with glutarald eh yd e a s describ ed by Azem et al . data for t h e velocity of th e ATPa se r eaction wer e fitt ed to th e Hill (1994 a ) confirmed that with in th e t im e sca le of th e ex pe ri me n ts eq ua t ion u sing the nonlin ea r r e gr essi on meth od of th e En zfitter com - described , > 95% of th e muta nt chaperonin wa s in th e tetra- puteri zed program a ccor d ing to Leath erbarrow ( 198 7 ). Th e following decam eric form (d ata not sh own ). Thus , th e ge ne ra l st ru ct u re cons ta n ts wer e derived (see T ab le I ): Ken' = Vma/ IGroE L J, K' = IS]".a" , of th e ch ap eronin wa s not affect ed by th e mutati on . a n d n Il = Hill coe fficie nt. Chemic al Cross -lin k ing of Chaperonin Olig omers-Th e bind in g of Geneti c Complem enta ti on wit h Wild-typ e and Mutant Gr oES to wild-typ e a n d mutant Gr oEL ,.• in solu t ion wa s m ea sured GroEL- In E . coli , expression of pla sm id -en coded (pCh Vl) lu - u sin g ch em ical cr oss -li n ki ng a t 3 7 °C wit h glutara lde hy de (0 .2 2%) and ciferase operon from V. [ischeri is u n der th e t r a ns cr ipt ion a l S DS -polya cry la m ide g e l e le ct r oph ores is as d escrib ed by Aze m et al . control of GroE ch ap eronin s (Ada r et al . , 1992 ). Thu s , wild -typ e (1994 a , 199 4b ). E . coli cell s harborin g pla smid pChVI emit 1Q4- 5_f old mor e Refolding of Rubisco'<In vitro ch a pe ro n in-d e pe n d e n t r efoldin g of visib le light in th e ea r ly stat iona ry phase than T Gr oEL Rubi sco from Rhodospirillum rubrum wa s assaye d at 25 °C as d e- S50 sc r ibe d by Gol oubinoff et al . (19 89 ). Rubi sco (20 IJ.M ) wa s den a tured in 4 mutant cells. Wh en E . coli T mutant cells h a r bor in g th e two S50 Murea a nd 10 rnxtdith ioth r eitol a n d th en di luted SO-fold into a so lu t ion comp atib le plasm ids pChVI a n d pTrc99A were s pot te d with T of 5 0 mxr T ri s , pH 7.5 , 20 mxt MgA c , 20 rnxr KCl , 1 m M dithiothreitol , bacteriophages , a plaque did not dev elop (F ig. 3A, cell la wn 2 ) 20 mxt g lu cose , 1 mxt ATP, 6 IJ.M Gr oES (p r ot orne r ), an d 3 IJ.M wild- t yp e and th e cell la wn emit te d very low level s of light (F ig. 3B , cell or mutant Gr oEL. Hexok in a se (S igm a) wa s a d de d t o a fin a l conc entra - lawn 2) . When , how ever, th e GroE-l ess pTrc99A pla smid wa s t ion of 40 u g/ rnl to s to p th e r efo lding r ea cti on a t th e ind ica te d tim e rep laced by pTrcESL, th e T cell lawn emitte d high level s of points . Rubisco a ct ivity wa s det ermin ed as d escribed by Goloubin off et S50 al. (1989). light (F ig. 3B , cell la wn 1) and dev eloped a plaqu e (F ig. 3A , cell In hibition of Protein Refoldin g-Pi g h eart mitochondri a l m a late d e- lawn 1). Wh en pTrcESL was r ep lac ed by pTrc87 containing hydrogena se (9 IJ.M m on om er; Boehringer Mannh eirn ), d en atured in 4.5 GroEL with t h e D87E muta tion , a n interm ediat e ph en otyp e ~I urea for 3 h a t 25 °C , wa s di luted 70 -fold into a r efoldin g so lu t ion was observed: th e cell la wn emit te d only low lev els oflight (F ig. contai ning in cr ea sing a m oun ts of mutant or wild- t yp e GroEL , 10 mxt 3B , cell la wn 3 ) but s uccess fu lly develop ed a T plaq u e (F ig. 3A , KCl, 20 mxi MgAc 5 0 mM tri eth an olam in e , pH 7.5, a n d 2 ms: dithio - 4 2, cell lawn 3) . Thus , th e m utant ch ap eronin is partia lly fun c- t hreitol. Ma late dehydrogenase a ct ivity wa s assayed at 25 °C in 150 mn K + pho sphate buffe r , pH 7.5, 0.5 mx: oxa lace ta te, 2 m M dithiothre itol , tional in viv o. and 0.2 mg / m l NADH by m ea surin g th e time-d e pendent ch a nge in t h e Ch ape ronin-ass isted Refolding of Rubisco in Vitro -When ab sorption of mon ochrom atic light a t 340 nm (Mill e r et aI. , 1993 ). com pa re d with wild -typ e GroEL , the chaperonin -dep end ent I4 Geneti c Complem entation of Phage Morph ogenesi s-The E. coli mu- r efolding of t h e Rubi sco e nzy me by mutant D87E ex h ibite d a tan t s t rai n TH aDca r ries a chrom oso mal mutation in th e groE L ge ne that slower r ate and r eached a maximum r ecovery of - 25% th at pr events maturation of A-bacterioph a ge prohead s (Taka no a nd obt ain ed with the wild-typ e chaperonin (F ig. 4 ). Kakefuda , 1972 ). T " wa s tran sform ed with e it he r pTrcESL or pTrcS7 , H a a nd p h a ge morph ogenes is wa s t ested u sing a "s pot test" for plaque I n h ibit ion ofSpontan eou s Prot ein Refolding-At 25 cC, urea- formation with T bacterioph ag e a ccor d ing to Reve l ( 19 80 ) a n d Gr een er denatured ma late dehyd r ogen a se can r efold s pon t a ne ous ly into et al . (199 3). native enzyme (Mill er et al. , 1993 ). In th e pr es enc e of GroEL , I4 Geneti c Complem entation of Lu ciferase Tran script ion-In E. coli , th e however, th e malate dehydrogenase folding interm ediates bind tran scription of a pl a smid-encod ed lumin escenc e (l ux ) operon from to th e chaperonin and are pr evented from r efold ing into an Vibri o [ischeri , pCh VI (Ul it zu r a nd Kuhn , 198 8 ), requi r es hi gh lev e ls of active enzy m e . Fig . 5 sh ows that th e concen tration of D87E Gr oEL a nd GroES (Ad a r et al. , 1992 ). Wi ld -t yp e E. coli cell s carryin g 4 5 pChV l e m it le ve ls of visibl e light 10 - times high er th an T H aD ce ll s GroEL required to inhibit h a lf the s pon t a ne ou s r efolding of l4 ca r ry ing pChVl (Ad a r et aI. , 1992 ). Ce ll la w n s of T carryi n g t h e eao m alate dehydrogenase is four tim es hi gher t han for wild-t ype comp atib le pl a smid pTrcESL or pTrc87 in a d d iti on t o pChV l wer e GroEL J4" Th is indic ates th at t h e spontaneo us pa ssi ve binding of nonnativ e protein is deficient in th e mutant. Stabi lit y of th e Binary Com p lex- Wh en wild -typ e GroEL l4 , T h e a bbrevia t ion u s e d is : Rubisco , r ibul ose -l ,5-bi sphosphate car- boxyl a s e/oxy genase . wa s fir st ch all enged with nonnative Rubi sco, incubated with 13958 GroEL Mutant in Protein Binding and ATP Hydroly sis A B ~ 1 0 0 J: ::E Cl c: 'C 6 0 Gl a: !II :::s Ql c: 11l FIG. 3. Genetic c omp lementation of a GroEL m utant h o s t 2 0 strai n. Th e E . coli Gr oEL mutant s t ra in T (Ta ka no a n d Ka ke fuda , 8 5 0 0 1972) was trans formed with a pla sm id , pGhV1 , con ta i ni ng th e lu cifer - CIl ase ope r on fr om V. fi scheri (U lit zu r a n d Kuhn , 1988 ) a n d with a com - patibl e pla sm id : wil d-ty pe pTrcESL (cell law n 1), G r oE -less control pl a smid pT rc99A (cell la wn 2), or mutant pT rc87 (cell la wn 3 ). Ba ct e ri al 4 5 0 2 3 lawns on LB a ga r plates we re s potte d wit h 3 p.1of T ', bacteriophage (l0" [Gr oE L]14/MDH Rati o plaque-forming u n it s/ml ), Aft er a 20-h in cu ba t ion a t 30 °G, th e bacteri al lawn wa s visuali zed for th e formation of a ba ct eri oph a ge pl aqu e in th e F IG. 5 . Inhibition o f s p onta neo us m alat e de hydroge nase re - li ght (A) and in t h e dark (B ). folding b y w i l d- type a n d mu t a n t D 8 7E GroE L. Ma late deh yd r o- ge nase (MDH; 9 P.M ) in 4 .5 M urea wa s diluted into a r e foldi ng solu t ion conta ini ng increasing concentration s of wild-typ e (e) or mu t ant D8 7E (0) G r oEL .... Ma late deh ydrogen a se a ct ivi ty wa s measured a s describ ed ::e e..... und er "Ma t e r ia ls a nd Method s" a fte r 135 min of r efolding a t 25 °G. e- Gl Gl a: >- ... Ql ~ 80 .!!! ..c o :::s Ql a: a: Gl o 60 !II III ..c Gl a: ~ 40 Ql 0 5 1 0 1 5 2 0 25 80 § 2 0 Ql Time (m in) a: FIG. 4 . Time cou rse o f Rubis c o r efolding b y wild-t ype a n d mu- t ant D87E G roE L. Rubi s co (20 p.M ) , den atured in 4 Murea , wa s dil u t ed 80 -fold into a refoldin g m ixture conta ining 6 p.M GroE S , 1 mMATP , a n d 20 30 40 50 60 0 10 3 p.M wi ld -typ e ( e ) or mutant D87E (0) Gro E L as describ ed under Tim e ( m i n) "Ma te rials a n d Method s ." Aliquots wer e r emov ed a t variou s t ime inter- va ls , and Rubisco a ct ivi ty was det ermi ned as desc rib ed by Goloub inolT F IG . 6. Rubis c o recovery after incubation wi t h or without et al . ( 198 9). T h e perc ent r ecovery is ca lculated r elative to th e a ct iv ity AT P . Ur ea -den atured Rubi sco wa s di luted as descr ibed in th e lege n d of of th e sa me conce n t ra tio n of n ati ve Rubi sco . Fig. 4 in th e pr esenc e of wild -typ e (. a n d e ) or mutant D87E (0 a n d 0 ) GroEL .... Binary comp lex es were in cub a ted for in cr ea s in g a mo u nts of ATP for in creasing amo u nts of time, and then supplemented tim e in th e pr es en ce (0 a nd e ) or a bse nce (0 a n d . ) of ATP pri or to t he with GroES to initiate re folding, no si gni fica n t loss of recov - a ddit ion of Gro ES (6 p.M). Th e Gro ES -de pe nde nt re foldi ng reaction was a llowe d to pr ogr ess for 15 min a nd th en wa s a rreste d with glu cose a nd ere d Rubisco wa s observed (F ig. 6 , e ). In contrast, when h exokina se a s in th e legend of Fi g . 4. Th e per cent r ecove ry is cal cu lated Ru bis co-bou nd mutant D87E GroEL was incubated with ATP fro m th e Rubi sco ac tiv ity re covere d wh e n ATP a nd Gro ES we re pr o- for increasing amo unts of tim e and th en s upp lemented with vid ed a t th e tim e of t h e Rub isco diluti on . GroES , a sign ifica n t time- dep endent loss of recov erable Rubi sco wa s obs erve d (F ig. 6 , 0 ). If, h owever , the initial incu- bation was carried out in the absence of ATP , no los s of recov - typ e GroE L . Mor eover, ATP was hydr olyzed by t h e mu tant in erable Rub isco activity was observed, either for wild -ty pe or a noncooperati ve m anne r . However, GroES inhibited ATP hy- mutant D87E GroE L (Fig. 6 , • an d D , re spectively ). Th ese dr olys is sim ila r ly in mutant a n d wild-type GroEL (Ta ble 1). re su lts further indi ca t e that the bin ding an d rebinding of non - Th is su gges ts t hat mutant D87E is deficie nt in t h e ability to n a t ive p r ot ein to t he chaperonin a re a ffect ed by t he D87E bi nd a nd h ydro lyz e ATP, but not in th e ab ilit y to bind GroES • mu t a ti on . B inding of GroES to Mutant D87E and Wild -typ e ATPase A ctivity-The r ate of ATP hydr olysis by m utant GroEL I..-The binding of GroES t o mutan t D87E or wild- typ e D87E is h alf that of wild-type GroE L (Fig. 7). Ta ble I sum - GroEL oligomers in the pr esen ce of ATP was meas ured usin g 1 4 14 marizes t he kinetic parameters for t h e ATPase activity of mu- che mical cro ss -linking wit h glutaral de hyd e a nd SDS-po lyacryl- tant and wild -typ e Gro EL • Th e affinity of AT P for mu t a n t amid e gel electrophore sis a s de scrib ed by Azem et al. (l994b ). 1 4 In th e presence of a saturating concentration of ATP , GroES D87E was nea rly a n or der of m agnitu de lower t han for wild- 7 OroEL Mutan t in Protein B inding and ATP Hy d roly sis 13959 7 0 C 1 2 3 4 5 67 8 . ) ... G> 5 0 :Gb"r ~HW6 ~~ ~ i )2: -0 ... GroELl 4 a. ... G> a. Ul ... G> D1 2 3 4 5 67 8 B 1 2 3 4 5 6 7 2 0 c: ... ::J I- :Gb" r~HW 6~ ~ ~P2 : Gro ELl4 0 4 8 12 16 Time ( mi n ) FIG. 7. Time -d epe n d ent h y d r ol y si s o f ATP b y w il d -typ e (e) and mutant (0) GroEL. F IG . 8 . B indin g of GroES to w il d -t yp e a n d m uta nt GroEL14' Wild-type GroE L •• (3 J.tM pr ot omer ) (A) or D87E GroEL (B ) was TABLE I 14 inc uba t ed wit h 1 mMAT P a n d in cr ea s in g concen tr ations of Gr oES . T he Chara cteristi cs of A TPa se activity of wild-typ e r a t io bet ween GroES a n d Gro E L pr otom er s was 0, 0 .125 , 0 .25, 0.50 , and m utant D87E GroEL 0.75 ,1.0, a n d 2.0 in lanes 1- 7, r esp ect ively. Wild-type Gro EL (3 J.tM ) (C) T he K ' a nd n il va lue s for wild-type and m ut ant D87E GroEL wer e the or D87E GroEL (D ) was incuba t ed wit h a n exce ss of GroES (6 J.t ~I ) a nd a ve ra ge of eight a n d s ix experi me n t s , respec t ively. Val u es for th e inh i- InCrM siri.g con cen t r a t ion s of A'1'P (0,1 ,3 , 5, 10, 50,100, a n d 500 J.tMATP b it ion by UroES of t.he UroE L A'I'Pase wer e t h e average of four exp er - in lan es 1- 8 , re spec tive ly). Cross-lin kin g of cha peroni n h et er o-oli- im ents . Ken' va lu es are from a r ep resen t a ti ve experi me n t i n whi ch t h e gome rs with 0.22% glutara lde hy de , SDS -polyacryla mid e gel electro- pu rifi ed mu t a nt D87E pr otein wa s devoid of det ect a ble degradation ph or es is , a n d Coomassie Blu e sta ining wa s accordi ng to Azem et at. produ ct s a s j udg ed by n a ti ve SDS gels (da ta not show n ). (1994a). Cooperat ivity Inhibition by J(O GroEL K ent GroES ( n il) recovery of Ru bisco by t he mutant, in th e pr esen ce of ATP a nd min - mM % GroES?, was - 25% t hat of t he wild-ty pe chap er onin (Fig. 4). Wild-typ e 6.0 26.1 := 7.1 1.80 := 0 .14 38 .8 := 9.0 Th e bin a ry com plex bet ween mutant cha pe roni n a nd D87E 3.2 227 := 6 1 0 .88 := 0 .10 50.8 := 4.0 Rubi sco was a lso less stable t h an the wild-ty pe bin a ry complex whe n inc uba te d for inc r ea sin g peri ods of t im es wit h ATP . Ma r ti n et al. (199 1) sugges te d th a t ATP hydrolysi s , in t he h a s t he sa me a ffin ity for wild-ty pe Gro E L (Fig. SA) as for 1 4 a bse nce of Gro ES, ca n ca use bound pr oteins t o und er go mu lt i- mutant D87E Gro E L (F ig. 8B ). However , in t he p res enc e of a ple fu t ile cycles of re lease a n d rebindin g to Gr oEL . Whil e t he saturati ng conce ntration of Gro ES ?, Gro ES bin di ng r equ ir es wild-ty pe bin a ry com plex a p pea re d t o wit hs ta nd multiple 5.7-fold les s ATP to bind wild- typ e Gro E L (F ig. sc: compar ed ATP a se-dr iven cycles of protein r elea se a nd re bi ndi ng wit hout with mu t an t D87E (F ig. 8D). Th e effective concen t rations of a significa n t loss of recover abl e Rubi sco, the mu t ant binary ATP neces sa ry for th e bindin g of Gro ES? to h a lf of t he Gr oEL com plex was signi fica n tly dest abilized by ATP hyd r olysis (Fig. oligome rs wer e 34 a n d 6 /L M for mu t a n t a n d wild- t yp e Gr oEL , 5 ), suggesting th at the r ebi nding of Rubisco to t he mu t a n t is re s pectively. Th is s uggests t hat fu nct iona l bindin g of Gr oES ? t o less successfu l t h a n to th e wil d-ty pe molecul e. Thi s dem on- Gro EL is not dir ectly a ffecte d by the D87E mutation , bu t st rates t hat, alth ough t he mu t ation is in t he ATP -binding rather th ro ug h t h e decr ea s ed a ffinity of t he mutant for ATP . pocket , th e AT P hyd r olysis mech ani sm is fu nctional an d is DISCUSSI ON capable of dri vin g r ever sibl e conforma ti on al cha nges in the In thi s wor k , t he h ighl y conserve d resi due Asp- 87 of GroEL mu tant chap er onin . Furthermo re , it confirms th at mutan t D87E ch ap er onin s was mutated t o glu t a mic ac id. In a Gr oEL -defi- is primaril y a ffected in its ability to init ia lly bind a nd to subse - cie nt E . coli host cell , pla smid-enc oded mutant D87E r est or ed qu ently r ebind nonnat ive pr ot ein s duri ng AT P-drive n cycles . ph a ge morph ogen esi s as efficien tly as wil d-ty pe Gr oEL , but not A ki ne tic a na lysis of ATP depend en ce curves of t he Gro E L t he ex pressio n of the lu x opero n , indi catin g t hat th e mutant ATP a se showe d th a t t he maxim a l ATP a se ac tivity of th e mu- chape ro nin is partiall y fun cti onal in vivo. Whil e under ext re me ta n t wa s 50% th at of t he wild ty pe a nd th a t t he a ffinity of ATP conditio ns, t he D8 7E mu t an t m ay be less stable in vit ro th an for mu t ant D87E wa s a n or de r of m agn itude lower t ha n for wil d-ty pe Gro E L , it n everth eles s asse mbled in t o a functi on al wild -t yp e Gr oEL Fen t on et al. (1994) showe d t hat two differ- 14 w oligomer with t he sa me a pparent st r uc t u re as wil d-t ype ent charge muta t ion s a t t he sa me positi on , D87K a n d D87N, Gro E L . Mor eover , mu t ant ch ap er on in D87E retaine d, a lbeit r esulted in a com plete loss of ATP a se activity a nd, conse - at various levels , a ll t he cha pe ron in func ti ons , thereby a llowi ng qu ently, of t he prot ein r efoldi ng ac t ivi ty a n d Gr oES binding a n a na lys is of t he r ela t ionship bet ween ATP h ydro lysi s , Gr oES ability. Th e effects of our D87E mu t ation as well as of D87K binding, p rotei n bindin g, a n d pr otein folding in t he olig omer. a n d D87N a re comp a t ibl e wit h t he st ruc t u re a na lysis fr om Th e sponta neo us form a t ion of the cha pe ro ni n-m a la te deh y- x-ray crystallography, wh ich pla ces Asp -87 in t he ATP-binding drogen a se bin a r y complex was four t imes less efficient for pocket of Gro E L (Br a ig et al., 1994; Fen t on et al., 1994; Kim et mu t ant D87E t ha n for wil d-ty pe Gr oEL indicating that mu- al . , 1994 ). Th e fac t that a s ign ifica nt level of pr ot ein r efoldin g tant D87 E h a s a lower a ffinity for nonn ati ve pr ot ein s . Cons ist- was a ch ieve d by a mutant la cking cooper a ti vity in ATP hydr ol- ent wit h t he 4-fold re ductio n in t h e a bility of the mutant cha p- ysis suggests t hat cooperativi ty of ATP hydrolysis may not be ero ni n to bin d nonn ati ve ma la t e deh ydrogen a se , the m axim al an a bso lute r equi r em ent of t he chape roni n -ass isted pr otein 13960 GroEL Mutant in Protein Binding and ATP Hydrolysis L., and Sigler, P. B. (1994) Nature 371, 578-586 folding mechanism, as previously suggested (Bochkareva et aZ. , Chandrasekhar, G. N., Tilly, K, Woolford, C., Hendrix, R, and Georgopoulos, C. 1992; Jackson et aZ., 1993; Langer et aZ., 1992). (1986) J. Bioi. Chem. 261, 12414-12419 Higher concentrations of ATP were required for the binding Cheng, M. Y., Hartl, F.-V., Martin, J., Pollock, R A., Kalousek, F., Neupert, W., Hallberg, E. M., Hallberg, R L., and Horwich, A. L. (1989) Nature 337, 620-625 of GroES to the mutant compared with the wild-type chapero- Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12,387-395 nin oligomer (Fig. 8, C and D). This can be explained by the Fenton, W. A., Kashi, Y., Furtak, K, and Horwich, A. L. (1994) Nature 371, 614-619 lower affinity of mutant D87E for ATP. A titration of the Frydman, J., Nimmesgern, E., Ohtsuka, K, and Hartl, F.-V. (1994) Nature 370, GroES-dependent binding of GroES to GroEL in the pres- 7 14 111-117 ence of a saturating concentration of ATP showed that GroES Goloubinoff, P., Christeller, J. T., Gatenby, A. A., and Lorimer, G. H. (1989) Nature 342, 884 - 889 has the same affinity for wild-type GroEL as for mutant Gray, T. E., and Fersht, A. R (1991) FEBS Lett. 292, 254-258 D87E GroEL (Fig. 8, A and B). This was confirmed independ- Greener, T., Govezensky, D., and Zamir, A. (1993) EMBO J. 12,889-896 ently when the same concentration of GroES inhibited half of Hemmingsen, S. M., Woolford, C., van der Vies, S. M., Tilly, K, Dennis, D. T., Georgopoulos, C. P., Hendrix, R W., and Ellis, R J. (1988) Nature 333, 330-334 the ATPase activity in the wild-type oligomer compared with Hendrick, J. P., and Hartl, F-V. (1993) Annu. Rev. Biochem. 62,349-384 the mutant oligomer (Table I). Furthermore, the binding of Hendrix, R W. (1979) J. Mol. Bioi. 129, 375-392 Higuchi, R (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, GroES to the mutant was functional since it ultimately re- M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) pp. 177-184, sulted in the refolding of Rubisco. Academic Press, Inc. San Diego, CA A cluster of GroEL mutants, all impaired in the ability to Hohn, T., Hohn, B., Engel, A., and Wurtz, M. (1979) J. Mol. BioI. 129,359-373 Horwich, A. L., Low, K B., Fenton, W. A., Hirshfield, 1. N., and Furtak, K (1993) bind nonnative proteins, was shown to be located in the apical Cell 74, 909-917 domain of GroEL, facing the upper central cavity of the oli- Jackson, G. S., Staniforth, R A., Halsall, D. J., Atkinson, T., Holbrook, J. J., gomer (Braig et aZ., 1994; Fenton et aZ., 1994). Remarkably, all Clarke, A. R, and Burston, S. G. (1993) Biochemistry 32, 2554-2563 Kim, S., Willison, K R, and Horwich, A. L. (1994) Trends Biochem. Sci. 19, the protein-binding mutants were also found to be impaired in 543-548 the ability to interact with GroES , suggesting that nonnative Kusukawa, N., and Yura, T. (1988) Genes & Deu. 2,874-882 Kusukawa, N., Yura, T., Ueguchi, C., Akiyama, Y., and Koreaki, 1. (1989) EMBO proteins and GroES compete for common sites in the inner J. 8, 3517-3521 face of the apical domain of the GroEL core oligomer (Fenton 1 4 Langer, T., Pfeifer, G., Martin, J., Baumeister, W., and Hartl, F.-V. (1992) EMBO et aZ., 1994). Our findings suggest that the protein-binding sites J. 11,4757-4765 Leatherbarrow, R J. (1987) Enzfitter Program, Elsevier Science Publishers B. V., are not necessarily located in the apical region of the central Amsterdam cavity, but may be located on the external envelope of the Lewis, V. A., Hynes, G. M., Zheng, D., Saibil, H., and Willison, K (1992) Nature GroEL cylinder, in the equatorial domain of GroEL (Braig et 358, 249-252 1 4 Llorca, 0., Marco, S., Carrascosa, J. L., and Valpuesta, J. M. (1994) FEBS Lett. al., 1994). This finding is of particular importance in view of 345, 181-186 recent observations that symmetric GroEL ) 2 oli- 14(GroES7 Martin, J., Langer, T., Boteva, R, Schramel, A., Horwich, A. L., and Hartl, F.-V. (1991) Nature 352, 36-42 gomers, in which both access ways to the central cavity are Martin, J., Horwich, A. L., and Hartl, F.-V. (1992) Science 258, 995-998 obstructed, are nevertheless fully functional chaperonins capa- Mendoza, J. 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