Proc. Natl. Acad. Sci. USAVol. 90, pp. 9828-9831, November 1993Biochemistry

Escherichia coli ribosomal protein L7/L12 dimers remain fullyactive after interchain crosslinking of the C-terminal domainsin two orientationsANDREW V. OLEINIKOV, GEORGE G. JOKHADZE, AND ROBERT R. TRAUTDepartment of Biological Chemistry, School of Medicine, University of California, Davis, CA 95616

Communicated by Daniel E. Koshland, Jr., July 6, 1993

ABSTRACT Cysteine site-directed mutagenesis was usedto create variants ofEscherichia coli ribosomal protein L7/L12that have single cysteine substitutions, at residues 63 or 89,located in different exposed loops in the structure of theglobular C-terminal domain indicated by the crystallographicstructure. That structure shows a possible dimer interaction inwhich the two sites of cysteine substitution appear to be toodistant for disulfide bond formation. After mild oxidation insolution both of the overexpressed purified cysteine-substitutedproteins formed interchain disulfide crosslinked dimers in highyield. Both crosslinked dimers were fully active in restoringactivity in poly(U)-directed polyphenylalanine synthesis to ri-bosomal core particles depleted of wild-type L7/L12. Theseresults show that the two C-terminal domains have independentmobility. The activity of dimeric L7/L12 does not require theindependent movement ofthe two globular C-terminal domainsin an L7/L12 dimer; moreover, it appears independent of theirmutual orientation when joined by crosslinking at the twoloops. A third variant with a cysteine substitution at residue 33near thejunction between the ax-helical N-terminal domain andthe flexible hinge was prepared and tested. This protein wasactive in the protein synthesis assay in the reduced state.Oxidation produced the interchain crosslinked dimer in highyield, but this crosslinked dimer was inactive in polyphenyl-alaine synthesis. The inactivation was due to the inability ofthe Cys33-Cys33 oxidized dimer to bind to the core particle.

Ribosomal protein L7/L12 of Escherichia coli is the mostextensively investigated representative of the small four-copy dimeric acidic proteins that are found in large ribosomalsubunits of all organisms. In eubacteria, eukaryotes, andarchaea, the acidic proteins always exist as a conservedquaternary structural element in which two dimers are inte-grated into the ribosome through binding to a commonanchoring protein, L10 in E. coli (1-3). One or both of theL7/L12 dimers forms a conspicuous morphological featureon the ribosome known in E. coli as the L7/L12 stalk (4). Theproteins can be simply and selectively removed from andrestored to the ribosome with the concomitant loss andrestoration of activity (5). In both eubacteria and eukaryotes,the proteins are required for the efficient binding of elonga-tion, initiation, and termination factors. This is a majorexample of ribosome function in which specific proteins playa clearly defined and perhaps dominant role.

Protein L7/L12 of E. coli is composed of two distinctorganized structural domains (2): an elongated helical N-ter-minal domain, residues 1-36, that is responsible for the strongdimer interaction (6) and for binding of the dimer to L10 (7)in the 50S ribosomal subunit and a globular C-terminaldomain, residues 53-120, responsible for interaction withfactors (8, 9). Truncated L7/L12 fragments that lack the

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C-terminal domain fail to support protein synthesis eventhough they bind to the ribosome (8) and antibodies to theC-terminal domain inhibit the binding of elongation factors aswell as protein synthesis (9). The two domains are separatedby a putative flexible hinge, amino acid residues 37-52 (10),considered to confer mobility of the C-terminal domainessential to its functional interactions (11).The high-resolution crystal structure of the C-terminal

domain of the protein has been determined (12). It has acompact plum-shaped tertiary structure, with a high contentof secondary structure, and contains three a-helices andthree (3-strands, arranged as (1a)A(a8)eB(a13)C (13). The crys-tal structure has a twofold symmetry axis that relates oneC-terminal domain to another through an extensive contact.The surface involved in dimer interaction from the E. colicrystal structure, which involves residues from both mem-bers of the dimer, contains a large number of conservedamino acid residues based on comparison with other pro-karyotic L7/L12 amino acid sequences. This led to theproposal that the conserved surface was involved in inter-actions with factors and implied that the two C-terminaldomains were arranged and functioned cooperatively on theribosome in the same orientation revealed in the crystalstructure (13).We have used cysteine site-directed mutagenesis (14) and

disulfide crosslinking to investigate whether the two C-ter-minal domains of an L7/L12 dimer in solution can makecontacts other than those indicated by the crystal structureand then whether dimers crosslinked in different orientationsare active in restoring activity in protein synthesis to ribo-somal core particles depleted ofwild-type L7/L12. Wild-typeL7/L12 has no cysteine residues. Cysteine residues weresubstituted at residues 89 and 63 in the C-terminal domain,and for comparison, a third cysteine substitution was made atresidue 33 near the junction ofthe N-terminal domain and theflexible region.

MATERIALS AND METHODSPreparation of Ribosomes, Cores, and Reconstituted Parti-

cles. Ribosomes were prepared from E. coli MRE600 asdescribed (15). Protein L7/L12 was selectively removed witha solution containing ethanol and NH4Cl to produce 70S P0cores (16). Reconstitution of ribosomes from Po cores wascarried out by mixing the cores with disulfide-crosslinked orcontrol L7/L12 in a ratio of 6:1 (a 1.5 equivalent excess ofL7/L12) and assayed directly for protein synthesis activitywithout isolation of the reconstituted ribosomes (17). Thepoly(U)-dependent polyphenylalanine synthesis assay wascarried out in 100 IlI as described (17), typically with 20 pmolof reconstituted particles and 120 pmol of various variants ofL7/L12. The L7/L12 was analyzed to determine whether itwas still present as the disulfide crosslinked dimer after theprotein synthesis assay by SDS/PAGE of the entire reactionmixture and Western blot analysis with a monoclonal anti-

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body to the N-terminal domain of L7/L12 (9). The binding ofL7/L12 after reconstitution of Po cores with Cys33 or Cys63was determined after centrifugation of the particles through10%o (wt/vol) sucrose in buffer A (20 mM Tris-HCl, pH7.5/100 mM NH4Cl/10 mM MgCl2) in the TLA rotor (Beck-man) at 100,000 rpm for 1 h at 4°C, resuspension in buffer A,and a second centrifugation. Samples of the purified recon-stituted particles were analyzed on a Western blot with themonoclonal antibody to the C-terminal domain ofL7/L12 (9).

Construction, Overexpression, and Purification of Cys89,Cys'3, and Cys3 Variants ofL7/L12. Genetic constructions forSer89 -* Cys and Ala63 -_ Cys substitutions have been de-scribed (17, 18). The Ser33 -_ Cys substitution was generatedusing the site-specific oligonucleotide-directed in vitro muta-genesis system from Amersham. The DNA sequence wasconfirmed using the Sequenase T7 system from United StatesBiochemical. All procedures for overexpression, purification,and analysis ofprotein variants have been described (18). Theproteins [Cys89]L7/L12, [Cys63]L7/Ll2, and [Cys33]L7/L12are designated Cys89, Cys63, and Cys33 hereafter.

Oxidation of Cysteine Variants of L7/L12. Pure proteinswere oxidized at a concentration of 0.2 mg/ml by 1 mMCu2+(o-phenanthroline)3 (14, 19) in the absence of reducingreagent at 25°C for 10-60 min. The oxidation reactions werestopped by addition ofEDTA to 50mM and blocking possibleunoxidized SH groups by incubation with 40 mM iodoace-tamide for 60 min at 0°C.

Analysis ofL7/L12 Proteins. HPLC gel filtration ofL7/L12before and after oxidation was carried out using a column(TosoHaas, G3000-SWXL, 30 cm x 7.8 mm i.d.) equilibratedin buffer A at a flow rate of 0.3 ml/min. Electrophoreticanalysis of the proteins was carried out with the PharmaciaPhastGel system in gels containing 20%o polyacrylamide. Fig.1 shows the purity of the three protein variants. Immunoblotanalysis of proteins after SDS/PAGE with anti-L7/L12monoclonal antibody was carried out as described (17).

RESULTSLocation of the Cysteine Substitutions in the C-Terminal

Domain. Fig. 2 is a drawing of the two C-terminal domains ofa L7/L12 dimer arranged as indicated by the crystal structure(13). Residue 89, where a Ser -+ Cys substitution was made,is located in the turn between the aB-helix and the PB-sheet,and residue 63, where an Ala -- Cys substitution was made,is in the turn between the PA-sheet and the aA-helix.Although the two Cys89 residues are on the facing surfaces ofthe two monomers in the crystal structure, the distanceacross the twofold axis appears too great to permit efficientintradimer disulfide formation (A. Liljas, personal commu-

1 2 3 4

A.

89- SH SH- 89

63- SH

FIG. 2. Simplified model of the two C-terminal domains ofL7/L12 taken from the crystallographic structure (12), showing thelocations of the site-directed cysteine substitutions.

nication). The distance between the two Cys63 residues is sogreat in the crystal structure as to totally preclude intramo-lecular disulfide bond formation in the arrangement depicted.

Disulfide Crosslinking of Cysteine Variants. Oxidation insolution of the three cysteine variant proteins was promotedby Cu2+(o-phenanthroline)3 and was monitored by SDS/PAGE. The results in Fig. 3 show that all three L7/L12variants were oxidized almost completely. The mobility ofwild-type L7/L12 did not change. Dimer formation wasabolished by addition of 6 M guanidine hydrochloride or 1%SDS. The rate of oxidation was rapid and nearly the same forthe three proteins. It is dependent on the concentration ofCu2+(o-phenanthroline)3 and in typical experiments was-50o of oxidized dimers formed within 30 sec at 15 uMCu2+(o-phenanthroline)3 and room temperature. Gel-filtration analysis (Fig. 4) of sample containing mixed oxi-dized and reduced Cys89 showed a slight increase in theelution volume of oxidized Cys89 compared to the reducedprotein, consistent with the formation of a more compactstructure by crosslinking. The same effect was observed forCys63, whereas Cys33 did not change after oxidation. Theresult demonstrates that the oxidation reaction forming thedisulfide crosslink was intramolecular and not attributable toan intermolecular reaction between two dimers. The forma-tion of disulfide-crosslinked dimers was also apparent whenribosomes that contained the variant proteins were oxidizedand then analyzed on a Western blot with an antibody toL7/L12 (results not shown).

Activity of Oxidized Disulfide-Crosslinked Dimers. Thethree oxidized L7/L12 dimers were tested for their ability torestore polyphenylalanine synthetic activity to Po cores de-pleted of wild-type L7/L12. The assay components con-tained none of the reducing agents typically present. Table 1shows the results with the reduced and oxidized dimers. All

FIG. 1. SDS/PAGE of purified L7/L12 protein variants used inthe experiments presented. Lanes: 1, Cys89; 2, Cys63; 3, Cys33; 4,wild-type L7/L12.

1 2 3 4

FIG. 3. SDS/PAGE of the L7/L12 variants after oxidation.Lanes: 1, Cys33; 2, Cys63; 3, Cys89; 4, wild-type L7/L12.

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2

1 2 3

Dimer _

Monomer -

FIG. 4. HPLC of oxidized Cys89. (Inset) HPLC fractions 1-3were analyzed by SDS/PAGE (lanes 1-3, respectively).

three reduced noncrosslinked dimers are fully active com-pared to wild-type L7/L12. Oxidation to give nearly com-plete formation of disulfide crosslinks had no effect on thecapacity of Cys89 and Cys63 to restore activity. Oxidationcompletely inactivated Cys33. To explain the absence ofactivity of oxidized Cys33, the effect of crosslinking on thebinding of L7/L12 dimers to ribosomal core particles wasexamined in the experiment shown in Fig. 5. The reconsti-tuted ribosomes were purified by centrifugation to separatethem from excess or loosely bound L7/L12, and the totalprotein was examined by SDS/PAGE and Western blotanalysis. Oxidized dimers of Cys63 were present, but Cys33was present in greatly reduced amounts compared to Cys63(lane 3). Crosslinking of the Cys33 residues makes the dimerunable to bind strongly to the ribosome. Western blot anal-ysis of Cys63 was also done on the incubation mixture afterthe protein synthesis assay. There was no reduction of theoxidized dimers due to mixing and incubation with compo-nents of the protein synthesis assay as shown in Fig. 6 (lane1 vs. lane 2).

DISCUSSIONThe results described above are summarized schematically inFig. 7, which depicts the two organized domains linked by ahinge region. Cysteine site-directed mutagenesis of the threeresidues (indicated 33, 63, and 89) has produced L7/L12dimeric proteins that all undergo rapid intramolecular disul-fide crosslinking. The figure depicts different orientations forthe reduced (native) and oxidized crosslinked forms for eachvariant. Crosslinking was shown clearly to take place withinthe L7/L12 dimer and not between two dimers. While mostrecent discussions of the structure of L7/L12 have assumeda parallel arrangement for the members of the dimer, thequestions of parallel vs. antiparallel and staggered vs. alignedhave remained unsettled (11, 20). The formation of zero-length disulfide crosslinks in the three homodimers, each of

Table 1. Activity of 70S ribosomes reconstituted from Po coreswith disulfide-crosslinked L7/L12 dimers formed by oxidation

Activity

L7/L12 Reduced Crosslinkedvariant added dimers dimers

Po 0.4 0.7Po + WT 10.1 7.0PO + Cys89 10.4 8.6P0 1.5 2.3Po + WT 10.0 10.3Po + Cys63 11.0 11.7PO 2.3 2.8Po + WT 14.0 14.1Po + Cys33 14.4 3.2

Results represent number of Phe incorporated per 70S particle per15 min in poly(U)-directed polyphenylalanine synthesis. WT, wild-type L7/L12. For crosslinked dimers, dimers were oxidized beforereconstitution and reducing agents were absent from the incubationmixtures.

1 2 3 4 5

FIG. 5. SDS/PAGE and Western blot analysis with a monoclonalantibody to L7/L12 were used to examine the purified ribosomesreconstituted with Cys33 or Cys63. Lanes: 1, molecular weightmarkers; 2 and 5, pure Cys33 and Cys63 oxidized in solution; 3 and4, ribosomes reconstituted with oxidized Cys33 or Cys63 and purifiedby centrifugation before analysis.

which contains a cysteine residue at a different location, onenear the a-helical N-terminal domain and two in the globularC-terminal domain, argues strongly for the parallel alignmentof the two monomers. The formation of the Cys33 crosslinkargues for their nonstaggered arrangement. If the amino acidresidues at position 33 are in the-putative coiled-coil struc-ture, the result indicates that the residues are facing eachother; alternatively, residue 33 may be in a more flexibleregion that is part of the putative hinge. The former alterna-tive is supported by NMR experiments that show that Ser33is in an organized structural region of the dimer (21). Amonoclonal antibody against an epitope destroyed by acleavage between residues 29 and 30 caused the release ofone of the L7/L12 dimers from the ribosome (9, 22). Inaddition it has been shown that Phe30 is involved in theinteraction with L10 (6) through which the dimers are at-tached to the ribosome. It is possible that the Cys33 crosslinkperturbs the structure of the region ofL7/L12 responsible forthe interaction of one or both dimers with the ribosome,either because of its direct involvement or due to an indirectdistortion of the binding region.The location of residues 63 and 89 in the model (13) based

on the high-resolution crystallographic structure is shown inFig. 2. Residue 89 is located in the turn between the aB-helixand the ,B-sheet, and residue 63 is located in the turn betweenthe ,A-sheet and the aA-helix. Neither cysteine substitutionhad any effect on the activity of reconstituted particles, evenwhen iodoacetamide or a bulkier crosslinking reagent (23) wasattached at these sites. Although the two Cys89 residues appearto be on the facing surfaces of the two C-terminal domains inthe crystal structure, they are separated by a distance greater

L7/L12 DIMERR

L7/L12 MONOMER-

1 2 3

FIG. 6. SDS/PAGE and Western blot analysis with a monoclonalantibody to L7/L12 were used to examine the total assay mixture forprotein synthesis that contained ribosomes and oxidized Cys63 afterincubation for 15 min at 37°C. Lanes: 1, ribosomal proteins aftertranslation assay; 2, oxidized Cys63 dimer; 3, reduced Cys63 dimer.

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B 63S - S63 89S - S89

BOTH ACTIVE

C 33S - S33

INACTIVE::DOESNT BIND

FIG. 7. Schematic summary of the results presented. (A) Rep-resentation of one reduced L7/L12 dimer attached to L10 andindicating (i) independent mobility of the two C-terminal domainsand (ii) the separation of the two Cys33 residues at the junction of thehinge and L10-binding domains. (B) Representation of the differentorientations of the C-terminal domains depending on whether theCys89-Cys89 or the Cys63-Cys63 disulfide crosslink is formed. (C)Distortion of the normal Cys33 region caused by the Cys33-Cys33crosslink.

than the disulfide bridge and could form a crosslink only ifthere was significant local mobility. The distance between thetwo Cys63 residues is so great in the crystal structure as tototally preclude disulfide bond formation. The finding that therates and extent of intramolecular-disulfide-crosslinked dimerformation for the Cys63 and Cys89 variants were comparableand independent of the location of the cysteine substitutionwas unexpected in relation to the crystal structure of theC-terminal domain dimer. The results show that the interac-tion indicated in the crystal structure is not a strong one andthat mobility of the C-terminal domains exists. This mobilitymay either be conferred by the flexible hinge region or existwithin the globular domains themselves and is sufficient topermit different orientations, including that trapped bycrosslinking. Time-resolved fluorescence measurements haveshown both local mobility of residues 89 and 63 in L7/L12dimers and global mobility of the entire C-terminal domain.Energy transfer experiments have shown no transfer betweenprobes located on residues 63 or 89 in the respective dimers,in contrast to residue 33 where efficient transfer takes place (B.Hamman and D. Jameson, personal communication). Theresult indicates that on average the two C-terminal domainsare distant from each other and is consistent with the mobilityindicated by the formation of different crosslinks. The oxi-dized Cys63 and Cys89 dimers must have orientations disparatefrom each other and from the dimer interaction surface evidentin the crystal structure (Fig. 7). On the basis of comparativesequence analysis Liljas et aL (13) proposed the functionalimportance of a conserved contiguous surface composed ofportions of both C-terminal domains in the crystal structure.This surface could not be maintained in both of the disulfide-crosslinked dimers, as emphasized in Fig. 7. We conclude thatthe two C-terminal domains of a dimer have considerablefreedom to move independently of each other, but that theycan be locked into disparate proximal orientations in closecontact with each other with retention of full activity, and thatthere is no obligatory specific functional arrangement orinteraction of the two C-terminal domains and no functionalrequirement for the two globular domains to move indepen-dently of one another.

There is indirect evidence that it is the C-terminal domainof L7/L12 that is required for the binding of factors to theribosome (8, 9). The factor binding site is on the body of the50S subunit at the base of the L7/L12 stalk (24). Traut et al.(25) have proposed that one or both L7/L12 dimers can existin a bent conformation in which the C-terminal domain isclose to the factor binding site, and we have demonstrated(17, 23) a site-specific crosslink from Cys89 to L10 and Lll,both located on the body of the ribosome near the base of thestalk and the N-terminal domain of L7/L12. Crosslinksbetween L7/L12 and elongation factors G (25) and Tu (B.Nag and R.R.T., unpublished results) have also been dem-onstrated; however, the location of the crosslinking site inL7/L12 was not determined. We conclude that there is afunctionally important contact between the C-terminal do-main and the factors. The present results raise questionsconcerning the mechanism and specificity of this interactionand whether it involves one or both C-terminal domains ofone L7/L12 dimer. Also unexplained is the requirement fortwo dimers for maximal rates of protein synthesis.We thank Nick Zecherle and Majid Mehrpouyan for early contri-

butions in the production and characterization of the L7/L12 vari-ants. This work was supported by the National Institutes of Health(GM 17924).

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