Eur J Biochem 1-73, 131 -135 (1985) ( FFBS 1985

About the specificity of photoinduced affinity labeling of Escherichia coli ribosomes by dihydrorosaramicin, a macrolide related to erythromycin Sylvie SIEGRIST, Nicole MOREAU and Frangois LE GOFFIC

Centre d'Etude et de Recherche en Chimie Organiquc Appliquee, Laboratoire du Centre National de la Recherche Scientifique, Thiais

(Received June 27/August 28, 1985) - EJB 85 0697

Photoactivation of the [3H]dihydrorosaramicin chromophore at a wavelength above 300 nm allows the covalent attachment of the macrolide antibiotic to the bacterial ribosome. Bidimensional electrophoresis shows that the radioactivityisniainly associated with proteins L1, L5, L6, L15, L18, L19, S1, S3, S4, S 5 and S9. When photoincorporation of the drug is conducted in the presence of puromycin as effector of ['H]dihydrorosaramicin- binding sites, a decrease in the labeling of most proteins is observed, except for LIX and Ll9, which are radiolabeled to a larger extent. These results allow us to speculate that L 18 and L 19 belong to the high-affinity binding site of rosaramicin antibiotic.

Photoaffinity labeling has become an important method for identifying functional sites on the Escherichia coli ribosome [l] and antibiotics such as puromycin and chloramphenicol have been used as tools for providing infor- mation on the peptidyltransferase center [2 - 41.

Although it has been a quarter century since macrolide antibiotics were reported to block protein synthesis [S] the mechanism by which they accomplish this inhibition has re- mained unknown. Erythromycin, the major representative of the macrolide group, binds on the large subunit ofprokaryotic ribosomes [6] and, among other assumptions, could be considered as an inhibitor of the peptidyl residue near the peptidyltransferase [7]. In contrast to the large number of studies concerning the mode of action of the macrolides, very little is known about the macromolecules involved in the structure of their binding site.

Rosaramicin (Fig. 1) is a 16-membered macrolide, whose activity is similar to that of erythromycin. This antibiotic possesses an aldehyde function, easily reducible to an alcohol group without loss of antibiotic activity; this led to the obtaining of a tritiated molecule, which was used to determine rosaramicin-binding parameters onto E. coli ribosomes, showing one high-affinity binding site (Kd = 0.16 x M) and multiple low-affinity binding sites [8, 91. Moreover, the antibiotic has an a,p-ethylenic y,S-epoxyketo system that offers a promising approach to photoactivated affinity labeling. This method, which eliminated the need for pre- paring reactive derivatives, has been recently used to label steroid-binding proteins and receptors [lo]. In a preliminary report [I l l we have shown that photoexcitement of ['H]dihydrorosaramicin allows its covalent attachment to E. coli ribosome proteins, mainly on L1, L5, L6 and S1. Recently components of the macrolide-binding site on the ribosome have been identified [ 121 using dihydrocarbomycin under different labeling conditions and showing L27 as the major labeled protein.

Cor~e~~onrit.121(.t,tz~[~ to S. Siegrist, Centre d'Etudes et de Rechcrches de Chimie Organique Appliqute, Laboratoire du Centre National de la Recherche Scientifique, 2 i 8. rue Henri-Dunant, F-94320 Thiais, France

In this paper we report on experiments directed towards exploring the general validity and applicability of this photoaffinity labeling procedure.

EXPERIMENTAL PROCEDURE

Buffers used included (A) 20 m M Tris/HCI (pH = 7.6), 6 mM Mg(OAc),, 100 mM NH4Cl; (B) 10 mM K2HP04/ KH2P04 (pH = 7.6), 100 mM MgS04, 100 m M KCI.

Materials

Kosaramicin was a gift from Unilabo (France). [3H]Dihydrorosaramicin (2400 Ci/mol) was synthesized according to Siegrist et al. [8] and stored at 4'-'C in dimethylformamide as a stock solution or in water as a diluted solution; its stability was checked before experiments by ultraviolet absorption at 240 nm and by dialysis experiments.

Tight-coupled ribosomes from E. coli MRE 600 were pre- pared by zonal centrifugation using the procedure of No11 et al. [13].

Anulysis of co valent binding

Different procedures have been used. Extensive dialyses were performed with a dialysis membrane as previously de- scribed [8] or with agarose gels [14] at 20°C in buffer A or B with only 10 mM NH4Cl. Filtration on Sephacryl S-200 gel was carried out at 20'C in buffer A with 10 mM NH4C1 [8]. Ethanol and C13AcOH precipitations were performed according to Cooperman [I S ] and Westphal [lo] respectively.

Photolysis experimcnts

A Philips SP 500-W lamp with high luminance and an oriented beam (maximum emission at 360 nm) was used. In all photolytic experiments the mixture (0.48 pM ribosomes and 2 pM antibiotic) was incubated for 10 min at 37°C in burfer B, then irradiated for 10 min between 30°C and 37°C in pyrex tubes placed 8 cm away from the light source as previously described [4].

132

1.2

I -

ul Ll c 0.8 4 Q c O

Q T

W 0.6

0.4

0.2

Subunits and RNA separation

The separation of ribosomal subunits was performed by analytical centrifugation on 5 - 20% sucrose gradients according to Gualerzi [16] in buffer A with 1 mM Mg(OAc), and 10 mM NH4CI. The rRNA analysis was performed by ultracentrifugation on a 5 -20% sucrose gradient in LiCl/ sodium dodecyl sulfate as previously described [4].

-

-

-

NMe2

HI -

-

Extraction ojthe rihasomal proteins atid f nio-rlimensional polyacrylamidc gel electrophoresis

After irradiation, the mixtures were concentrated by poly(ethyleneglyco1) precipitation [I 71. After centrifugation, the pellets were resuspended in buffer A with 33 mM M ~ ( O A C ) ~ . The ribosomal RNA were precipitated by addi- tion of two volumes of acetic acid and, after 1 h at 2°C they were pelleted by centrifugation at 3000 x g. The proteins from the supernatant were precipitated by the addition of five volumes of acetone, left overnight at - 20°C and pelleted by centrifugation at 12000 x g [I 81. The pellets were rinsed three times with acetone, dried and resuspended in 8 M urea. The proteins were separated by bidimensional polyacrylamide gel electrophoresis [I 91 : the first-dimension separation was run either at acidic (system A) or at basic (system B) pH, separa- tion in the second dimension was carried out at acidic pH. Proteins were stained with Coomassie blue. The sliced electrophoresis gels were burned in an IN 4101 Oxymat (In- tertechnique) oven before determining their radioactivity by liquid scintillation counting.

RESULTS

Photolysis expc+nent.s

Fig. 1 shows the change i n the ultraviolet spectrum of a solution of rosaramicin as a function of irradiation time. A first-order plot of In AZ4() (0)-ln A240 (m) versus time allows the calculation of a t l j 2 of 10 min for the destruction of the chromophore under conditions paralleling those of a photoincorporation experiment.

Dunonstration qf a covalent complex and efTectors o f the photolabeling yield

The covalent complex formed during irradiation can be isolated by filtration on an exclusion gel (Fig. 2). The ratio counts min- ' A & gives a fourfold increase between experi- ments A and B. Fig. 3A shows that irradiation allows the incorporation of radioactivity in acid-precipitable ribosomes. The incorporation reaches saturation as a function of [3H]dihydrorosaramicin concentration. The ratio, covalently bound rosaramicin/reversibly bound rosaramicin, calculated at a total of 10 pM antibiotic concentration where only the high-affnity binding site is involved, allows us to determine the yield of the covalent reaction ( 1 % ) . In our search for effectors to optimize this low yield wc have found that buffer B [9] is more appropriate for the photochemical reaction (yield: 4%). Argon stream, low temperature, use of the whole ultraviolet range, NaRH, reduction and ethanol precipitation procedure do not affect the yield of the reaction. As expected, rosaramicin decreases the amount of radioactivity in- corporated. Fig. 3 B shows that [3H]dihydrorosaramicin in- corporation into ribosomes is light-dependent and an approx- imate half-life for the photoincorporation of [3H]dihydro-

1.4 1

I

200 240 280 Wavelength fnm )

Fig. 1 . C/zrrngcs in tlw ultraviolet sprctrum qfrosaramicin us u fiinc,tion qf' irradiation firne. Rosaraniicin in aqueous solution (0.1 mM) was irradiated for the times indicated. A f I l 2 for photolysis was obtained by a first-order plot of A240 (insert)

'i

Fraction number

Fig. 2. Filtration of [ Hldihtihgdrornsaramicin-ribosome c ~ o r n p l ~ ~ s c . ~ on S~plrucrtihgl S-200 gel. (A) Control without irradiation. (B) The mixture was submitted to irradiation (20 niin). Fractions of 1 in1 were collected and 200 pl of each fraction were counted. (0) Absorbance 260 nm, (0) radioactivity. 144 pmol7OS ribosomes were used for the filtration experiment

133

15000

10000

5ow

1 .--L _1_.1 I I

0 10 20 35 50 80 100 0 5 10 15 20 30 40 13H I dihydrorosaramicin IyMI Irradiation time ( m m l

Fig. 3. Pkotolub~ling qf7O.Y r.ihosonzc~s by I3 H]dihyc~rrorosuramicin. (A) Influcnce of[3H]dihydrorosaramicin concentration. ( A , ) The incubation mixtures (0.1 ml) contained ribosomes (0.48 pM) and [3H]dihydrorosaramicin as indicated and thcy werc submitted to a 10-niin irradiation; ( A 2 ) without irradiation; (A3) = (Al)-(A2) gives covalent binding. (B) Influcnce of irradiation time. (B,) The incubation mixtures (0.1 nil) contained ribosomes (0.48 pM) and ['HH]dihydrorosaramicin (20 pM) and they wcre submitted to an irradiation time as indicated; (B2) without irradiation; ( B 3 ) = (Bl ) - (B2) gives covalcnt binding. The radioactivily incorporated rcprcsents the amount of antibiotic retained on nitrocellulose filters after CC1,AcOH precipitation of the niixturcs. 48 pmol 70s ribosomes were used for each CCI,AcOH precipitation

A l

Fig. 4. Ti~,o-u'in~ensional polyucrj~lurnide gcl c.lectrophoresis labeling put tcm qf proteins derived f r om 70s ribosomcv irrudiaied with [3H]u'ihyu'rir.orosararnicin (system A ) . (A) Proteins from 30s subunit; (B) proteins from 50s subunit. (-----) Background of the polyacrylamide gel. S , - S 1 6 and Szo- LZ6 arc not scparated. 4.8 nmol 70s tight ribosoincs werc used for photolabeling experi- ments

rosaramicin can be obtained from a time-dependent study (f1,2 = 5 min).

Loco liza t ion of tlir photo la beling into ribosomes

Analytical centrifugation on sucrose density gradients of ribosomes and of rRNA shows that both ribosomal subunits are labeled by [3H]dihydrorosaramicin and that there is no ribosomd RNA labeling (results not shown). The determina- tion of the radioactivity distributed among the ribosomal proteins separated in the bidimensional electrophoresis system B demonstrates (Fig. 4) that radioactivity is mainly

associated with proteins L 5 > L6 > L1 and, to a lesser extent, S3 > L33 > S 9 > L19 3 S 4 > L32 > S7 > L18 3 S 14. The same experiments, carried out with two different puromycin concentrations, give a similar labeling pattern (Fig. 5). But, as can be seen in Table 1, the labeling of most proteins is strongly decrcased, except for L 18 and L 3 9, whose labeling is increased: 200% and 125% respectively. In spite of the lesser resolution of bidimensional electrophoresis sys- tem A, the system enables us to analyze other proteins and revcals S1 to be the most labeled protein (Fig. 6). Results similar lo those of system B have ben obtained but not exactly with the same order of labeling (either because of bad re- solution of some proteins. or because of differences in the yield of protein migration or in the lability of the covalent linkage): S1 > L18 > L1 > S9 > L 5 3 L6 > L19 > S 5 > L 15 > S 3 3 S4. Puromycin induces a strong decrease of all labeled proteins (Fig. 6) except for L15, whose labeling is only slightly affected (85%) , and for L18 and L19, whose labeling is unchanged and increased respectively (Table 1). As expected rosaramicin (8 pM) approximately halves the ribosomal protein labeling (result not shown).

DISCUSSION Our previous studies, concerning reversible binding of

rosaramicin on tight E. coli ribosomes, enabled us to deter- mine the antibiotic concentration range where it was possible to minimize low-affinity binding [8]. Under irradiation at a wavelength higher than 300 nm, photoincorporation of 13H]- dihydrorosaramicin itself into ribosomes was possible and the half-life for the photoincorporation of [3H]dihydrorosarami- cin into ribosomes is approximately the same as the t I i z for chromophorc photolysis (1 0 min), which allows us to demon- strate the chromophore dependence of light-dependent incor- poration or [3H]dihydrorosaramicin. We had previously shown [4] that, after a 10-min irradiation (2 x lozo quanta/ ml). 90% of the initial ribosomal activity was retained. Sub-

134

Table 1. Influmce qj‘puroinycin on /3H]dili.)?drarosaramicin pliotoincorporation onto ribosomal proteins The results are exprcssed, for each protein, as a percentage of radioactivity obtained froin Figs. 5 and 6 (Figs. 4 and 6 as controls). Only thc significantly labeled proteins have been considered for the calculation

hlectro- Puromycin L1 LS L 6 L I S L18 L19 L32 L33 S1 S3 S4 S S S7 S9 S14 phorcsis coiicii system

mM Yo ~ _ _ _ _ _ _ _ _ _ _ ~ _ _ _ ~ _ -__

A 0 1 “ 70 90 8 5 - 120 125 0 0 - 3s 75 - 4s 50 0 - 20 15 - 0 20 0

B 0 2h 55 35 40 85 9.5 140 - - 40 SO 65 40 - 50 -

0 2 b 65 3s 4s - 200 12s 0 0

Concentration leading to a 60% increase of macrolide binding ‘ Concentrdtion leading to d 100% increase of macrohde binding

A1 I

10W I l l

_- - --- 0 - 5 3 4 5 7 8 9 ll12D1~616171819iV~

Fig. 5 . nL.o-diri~c.rrsionol polwcrylainide gel c&ctrophoresis labeling pottwii of‘ proteim derivcd f iwn 70s ribosomes irradiated with ( 3 H ] d i h y ~ r ~ ~ r o . ~ ~ r ~ i m i c i i i (system A ) in tlie presence of ( A ) 0.1 in M ui?d ( B ) 0.2 inM puroni~.”~in. (1) Proteins from 30s subunit, (2) pro- teins from 50s subunit. (-- --) Background of the polyacrylamide gel

sequent ribosomal protein analysis by bidimensional electrophoresis was done using two different systems for complete identification of the proteins [19]. The measurement of the radioactivity associated with the proteins from the two systems shows that thc labeled proteins are L 1, L 5 , L 6, L 1 5 , L18, L19, S1, S3, S4, S5, S9 and to alesser extent L32, L33 and S7. All the radioactivity was found inside the stained spots, which is an important condition for the validity of our results. As the high-affinity binding only occurs at the 50s subunit, it is surprising to observe the labeling of some 30s subunit proteins. This could be related either to a non-specific labeling or to a labeling at the subunits’ interface.

3000

Fig. 6. TMwdimensionul polyacrylaniide gel electroplioresis labeling pcittern of proteins derived .froin 70 S rihosotnes irrurlicited tritli I3 H/di~~~vdroroscirrrrniein (sqstern B ) in the uhsence or in tlie prcsmc~c~ qf0.2 niM puroniycin. (A) Proteins from 30s subunit; (B) proteins from 50s subunit. Square hatching. increase of labeling or, vertical hatching, decrease of labeling induced by puromycin. (-- --) Background of thc polyacrylamidegcl. S16-Sl7, L27-L28, L32- L33, S12-L20, S20-L26 are not separated. 4.8 nmol 70s tight ribosomes wcre used for photolabeling cxperimciits

By comparing our results with the litcrature data dealing with the macrolides-binding site, the labeling of L15 is in agreement with the binding of erythromycin to purified L 15 alone [23]. To our knowledge, nothing has been published about interactions between the macrolide-binding site and proteins L1, LS, L6, strongly labeled in this study, but it is noteworthy that L 1 is located near the puromycin-binding site and the peptidyltransferase center [24]. We do not observe

135

labeling of L4 [25], L22 [26], S5, S 2 proteins [27] o r 2 3 s RNA [28] macromolecules involved in erythromycin resistance, but the loss of the ability to bind an antibiotic by ribosomal mutants does not imply the presence of the modified protein at the antibiotic binding site.

In order to determine which of the labeled proteins belong to the high-affinity binding site we performed labeling experi- ments with [3H]dihydrorosaramicin in the presence of puromycin. Indeed, we have previously shown that puromycin induces a 100% stimulation of L3H]dihydro- rosaraniicin binding [9], explained by a twofold increase in the affinity constant of [3H]dihydrorosaramicin to its receptor, without a change in the number of the high-affinity binding sites, [29]. Moreover a disappearance of low-affinity binding sites was also observed. We took advantage of these two findings to use puromycin in photolabeling experiments as a revealer of the high-affity binding site and as a scavenger of low-affinity binding sitcs. The two different puroinycin concentrations (0.1 mM and 0.2 mM), used in the experi- ments, led to a 60% and 100% increase of the macrolide binding respectively [9]. Thus the dramatic effect of puroinycin on photolabeling experiments: decrease in the labeling of most proleins, increase Tor L 18 and L19, supports the notion that [3H]dihydrorosaramicin photolabels the 50s subunit proteins L18 and L 19 from the higher-affinity site whereas the other L and S protein labeling occurs either from solulion or from lower-affinity sites. As expected from binding studies [29] dihydrorosaramicin and puromycin covalently bind to ribosomes at two distinct primary binding sites, since L23 and S 14, the major proteins labeled by puromycin [2, 301, are not labeled by dihydrorosaramicin. Moreover, we can also conclude that puroinycin does not modify the localisation of high-affinity binding site of dihydrorosaramicin since no new proteins appear to be labeled in the macrolide photoaffinity experiments performed in the presence of puromycin.

Our major labeled proteins and, in particular, L18 and L19 are never lebeled by antibiotics, such as tobramycin aminoglycoside 1311, tetracyclines I321 and chloramphenicol [3,4], without influence on the rosaramicin high-affinity bind- ing [9].

Assignment of L18 its being at or near the peptidyltransferase center must be correlated to the possible binding of rosaraniicin to the ribosomal P site [29] and to the influence of macrolides on the peptidyltransferase reaction. Thus, L I8 is labeled by two aryl azide derivatives of peptidyl- tRNA [33, 341. L18 has been shown to be a protein essential for peptidyltransferase activity in single omission/reconstitu- tion experiments [35]. Finally, in the consensus model of the 50 S ribosomal subunit, from Prince et al. [24], L 18 is located in the central protuberance, at the peptidyltransferasc center, near the puromycin-binding site and near L27, the protein also involved in the macrolide-binding site [12].

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