Tetracyclines induce changes in accessibility of ribosomal proteins to proteases

Biochimie ( 1%&j 78. 868-873 @ So&t6 fran;aise de biochimie et biologie mokulaire

ce c

/ Elsevier, Paris

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IV Kolesnikov, NY Protasova, AT Gudkov* iwitute

of Protein Research, Russian Academy of Sciences, 142292 Pushchino. Moscow Region. Russiu (Received 18 March 1996; accepted 1 July 1996)

Summary - Limite:i proteolysis was used to test the interaction of tetracyclines and some of their derivatives with ribosomes. Proteolysis of the free ribosomes was compared with that of the ligand-bound ribosomes. The interaction of different tetracyclines with ribosomes depends on their chemical structure and produces both a protective effect and an increased susceptibility to proteases of some ribosomal proteins in the 30s and SOS subparticles. Most of the proteins affected by tetracycline action are located on the head of the 30s and interface side of the 50s subunits. On the grounds of the obtained data one of the antibiotic-binding regions can be located near the ribosomal peptidyl transferase center. The effect of possible conformational changes induced by tetracyclines on the translation process is discussed. tetracyclines I ribosorxves / elongation factors / proteolysis

Introduction For several decades tetracyclines,

antibiotics with a broad range of activity, were Iwidely used against different Grampositive and Gram-negative bacteria [I, 21. There is evi-

dence that tetracyclines inhibit growth of bacteria by interaction with ribosomes. It was shown that ribosomal 30s subunits bind tetrac~yclinesand that ribosomal proteins S3, S7 and S14 are invoked in this binding [3,4]. Furthermore, the changes were found in the reactivity of some nucleotides (8892, U1052JYO54) in the 16s ribosomal RNA in response to tetracyckue binding [5]. Inhibition of aminoacyl-tRNA binding to sibosomes is the most evident functional effect of tetracyclines [6]. Nevertheless, many details of tetracycline interaction with ribosomes are still unclear. Proteolyticdigestion is nown as a sensitive probe to follow the conformational changes in proteins and ribosomes during their mm&ion with ligamds[7-91. Using a set of proteases with different specificity we have tested the interaction of tetracyclines with the 70s ribosome and their subunits. The results of proteolysis of ligand-bound ribosomes in comparison with those of free ribosomes are presented in this paper. Materials and methods Enzymes

Trypsin (EC 3.4.21.5), thcrmollysin (EC 3.4.24,4) and subtilisin (EC3.4.21.14) were purchased from Sigma (USA), ficin (EC 3.4.21.6) was

3.4.2 1.7) were from Boehringer-Mannheim (Germany), endoproteinase Glu-C of S aweus VS (EC 3.4.2 1.19) was from Promega (USA). [“HI-GMPPNP and [ “HI-GTP were from Amersham (UK). Rihosomes 70s ribosomes were prepared according to the published procedure [ 101and washed once by precipitation through a 20% sucrose cushion. Conrplex r.# rihosomes with elongation factor G (EF-G) The complex of ribosomes with EF-G and the uncleavable analog of GTP (GMPPNP) was prepared and checked according to the published procedure [I 11.The ribosomal complex with EF-G and fusidic acid was prepared using labeled GTP as described [ 121. Proteolytic digestion The proteolysis of ligand-free ribosomes was carried out in 40 mM Tris-HCI buffer (pH 7.6-7.8) with 50 mM ammonium chloride, IO mM magnesium chloride and 1 mM dithiothreitol (DTT) at 37°C. The following ratios of the enzymes to the total ribosomal protein in the reaction mixture (100 pL, 1 mg of ribosomes) and incubation time were respectively: I: 1000, 1:500 and 1:300, 15 min for trypsin; 1:60 and 1:40, 15 min for thermolysin; I :30, 30 min for chymotrypsin; i:30, 30 min for protease V8; 1:30, 15 min for proteinase K; 1:30, 15 min for subtilisin. In the case of thennolysin the buffer contained calcium chloride (5 PM). Proteolysis of ribosomes with antibiotics and EF-G was carried out as above after incubation of their mixture at 37 “C during 15 min. The concentration of antibiotics was usually in the range from 0.1 to 1mM. The antibiotics used in the study are listed in figure 1 and were a gift from the American Cyanamid Company.

from Serva (Heidelberg,Germany),elastase (EC

3.4.2 1.36). chymotrypsin

(EC 3.4.2 1.1). proteinase

*Correspondence and reprints

K (EC

Electrophoretic awalysis After proteolysis the reaction mixture was treated with two volumes of acetic acid to extract ribosomal proteins and the protein

869

CONH;! CN

61

CONkI Ii

H

H H

H

H CONHz

H

Fig 1. Tetracycline derivatives used in the study.

samples were analyzed by two-dimensional electrophoresis in system IV developed by Madjar et al [ 131. The results of proteolysis were estimated in terms of disappearance (or displacement) and decrease in the size of protein spots in the Coomassie R-250 stained gels as published in reference 191.

Results and discussion Proteolysis of &and-free

rihosomes

Figure 2 represents an example of electrophoretic patterrs of ribosomal proteins from protease treated ribosomes. Tht: results are summarized in table I. It is striking that most

ribosomal proteins are not cleaved with proteases, even under severe experimental conditions (a high enzyme-toprotein ratio, elevated temperature and prolonged time). Of the specific proteases, trypsin and glutamic acid-specific protease VS cleave five to six proteins. Only non-specific proteases, such as proteinase K, subtilisin and pronase, digest 13 to 15 proteins from a total of 52 in the E coli ribosomes. These results suggest that ribosomal proteins are quite tightly packed in the ribosome and that most of them have a well organized tertiary structure. Among 30s ribosomal proteins cleaved by proteases, most of them (S2, S3, S7, SM, Si3, SW) are located on the ‘head’ of the 30s subunit and near it (S5, S6, S 18). It is generally believed that the 3OS subunit of the E coli ribo-

4 ”

I

Fig 2. Two-dimensional electrophoresis of 70s ribosomal proteins. Thermolysin treated 70s ribosomes without the antibiotics (A), and with O-DMG-DMDOT (B). Arrows indicate the proteins sensitive to protease.

Table I. Proteins digested by a set of proteases in ligand-free E co11 ribosomcs.

s2 s3 S5 S6 s7 Sl3 Sl4

Sl8 SI’) S2l LX12

++++ ++++ ++++

++++ ++++

++++ +++

++++ +++

++++

+++

++++ ++++

+

++++ ++++ +“I-++

+++ +

++++

++

++++ +++

++

++++ ++++ +-t++ ++ ++++ -

++ +++ +++ +++ + + +

++++ +-l-+-t ++++ ++++ c++ + +

-I-++

++++

+++ +++ +++ ++ ++

++++ +++ ++++ ++++ +++

kY7 ++ L23 ++-I-+ L27,L28 +++ L30 ++++ ++++ -__I-.-I-____ --~~--~-++++. protein is digested completely: +++, a trace of protein can be seen; ++, protein is cleaved by about 50%; +, noticeable cleavage; -Y protein is not cleaved.

All the proteases listed in table I, with the exception of ficin

and elastase, were used in the ex Differences were observed bet of the free and ligated-bound ribosomes in the cases of molysin (table II) and chymotrypsin. Thermolysin cleaves eight proteins (table II) i ribosomes. In this case 9-D

cycline

the interaction of ribos

derivative

is 9-DMG-DMDOT. It acts at a ie at a 50: 1 molar ratio of the osomes. The strong inhibition on translation [ IS] correlates wel against proteolytic digestion. It is interesting that tetracyclines with substituents at carbon 7 (like 9-DMG-minocycline) have no protective effect on thermolysin digestion of ribosomal proteins, though 9-DMG-minocycline strongly inhibits poly Phe synthesis (V Shirokov, personal communication). 9-DMG-DMDQT, a drug without substitution at carbon 7, can compete 9-DMG-minocycline for binding with ribosomes whe concentrations of these antibiotics in the mixture were and 0.75 mM, respectively. The 9-nitro-minocycline does not compete with 9-DMG-D It seems that different substituents at the tetracycline’s carbon 7 or 6 can influence conformational changes of the drugs due to their hydrophobic (or hydrophilic) interaction with ribosomal components. This can lead to different steric

S 19 to the binding of tetracyclines was suggested by different approaches (see for review [ i,2]). In our work we have found also that some of the 5OS subunit proteins, such as L7/Ll2, L17 and L23, can be protected by antibiotics against the protease action (in the case of thermolysi:?), so this may also have a relation to the antibiotic binding. here are data that tetracycline stimulates cross-linking uromycin to protein L23 [ 181. Puromycin is a well n antibiotic which interacts with the ribosomal peptiI-transferase center. Recent cross-linking studies of the ~o~~yci~ to ribosomal components without tetracycline ealed the L23 proteirl among the proteins crossromycin; however, the L29 protein was identified which is a close neighbor to L23 and authors did not exclude that L23 is exposed near or at the peptidyltransferase center in the 78s ribosome [ 191. Recent data of streptomycin on ribosome function also permit to place L23 near the peptidyltransferase center [20]. The modern morphological models of ribosorne with hollows. ledges and tunnels [21.22] can help to explain the discrepancy in location of L23 with previous results 1231. Our data on tetracycline’s protective effects on proteins located on the head of the 3OS subunit (S3. S7) and L23 of

Table II. Proteins digested by thermolysin in 70s ribosomes with antibiotics (electrophoresis results). Protein

:3 S6

70s withow antibiotic

70s +DMG DMDOT

70s + tetrucylirr

++++ ++++ ++++ ++++ ++++ ++ ++ ++ +++ ++++ ++++ ++++ ++++ ++-I-+ ++++

++ ++++ ++++ + ++++ + ++ f +++ + --*Concentration of antibiotics; + and - signs are explained in

;:* L7u.412 L17 L23

+++ .++-I-+ ++++ ++ ++++

++++ -

+ ++++ +-I-+ + +++

-__ 7OS+ DMDOT

705 +N,9-di-t-hlr~yl DMDOT

++++ +++ ++ ++++ ++++

++++

++ ++++ ++ + +

legend to table 1.

++++

+++ ++++

+ +++ ++++ ++++ +++ ++++ + ++ +++ +++ _~.__.__.___-

.___-._ 70s + c*pc~noc~hh~~~ rrtrm y fin

++++

+++

++++ + ++++ ++++ ++ ++++ __..-- - ~-- -- -

872 the SOS subunit, the inhibition of aminoacyl-tr?.NA binding by tetracycline 163,as well as the stimulating effect of tetracycline on puromycin cross-linking to protein L23 [ 181permit us to believe that one of the tetracycline binding sites can be located near the ribosomal peptidyltransferase center. Other antibiotics, such as erythromycin, streptomycin, puromycin, kirromycin and chloramphenicol tested in this work do not reveal any effect on digestion patterns of ribosomal proteins. Proteolysis of ribosomal complexes with EF-G Two different complexes of ribosomes can be prepared with EF-G in vitro. The first with the uncleavable analog of GTP mimics the prehydrolysis stage of ribosomes in the ribosomal cycle [ 111. The second with GDP and fusidic acid (FA) imitates the posthydrolysis stage [ 121. The formation of these complexes has been tested with [WI-labeled GTP derivatives by the filter technique. The tetracyclines do not interfere with EF-G binding to the ribosomes. The yield of complexes was in the range of 70-90% with or without antibiotics. At the same time proteolysis of the ribosomal complexes with W-G reveals a new effect of the tetracycline action. In this case digestion of the S7 protein is remarkably increased by trypsin (table IIIA) and protein S I3 is protected (compare table I and table IIIA) Only a protection effect is observed with thermolysin (table BIB). AS known, factor G triggers some conformational changes, so that the accessibility of some proteins to proteases increases [9]. In our case tetracycline induces additional distortions in the ribosomal complex with EF-Cl and the S7 protein is cleaved to a greater extent by trypsin.

Recent studies show that ribosomal protection against tetracyclines is accomplished by expression of genes for TetM, TetO and TetQ proteins (see [ 1,2] for review) which have high homology to elongation factors Tu and 6 [24,25]. These Tet-proteins can remove the in ibitory effects of tetracyclines. Though the function mechanism of these proteins is unknown there is an assumption that TetM can act as a tetracycline resistant special factor [25] and it was shown recently that TetM protein can release tetracycline from ribosome [26]. The increased sensitivity of S7 protein to proteolysis after tetracycline binding, change of the accessibility of S6, L 17 proteins to protease (table III) and the stimulating effect of tetracycline on puromycin cross-linking to protein L23 1181 permit to suggest that in the process of protein biosynthesis tetracyclines might produce (or prevent in some cases) conformational changes in the ribosomes at different stages of the ribosomal cycle and that the translation factors and Tet-proteins are involved in this process. This generalized point of view could explain the lower inhibitory concentration of tetracyclines on the translation process [ 15 ] than in the case when one particular stage of the ribosomal cycle is investigated.

A&now

ts

We are thankful to Dr Y Gluzman, the American Cyanamid Company for a supply of tetracycline derivatives and encouragement and to Prof A§ Spirin for initiation of the work and critical reading of the manuscript. This work was supported in part by Grant 9404- 1205 1 from the Russian Foundation for Fundamental Research.

Table III. Proteolysis* in ribosomal comlexes with G factor in the presence of 9-DMG DMDOT (AB).

Protein

Rs+AB+FA

RS + GMPPNP+ G

RS+AB+ GMPPNP+ G

RS + FA + GTP + G

++++

++a +-“++ -+++

++++

++++ ++++

++ +

RS+ABi-FA+ GTP+G

A. Trypsin (protease/total Rs protein ratio -l/300, 15 min)

s7 s13 L7/Ll2

+ ++++ +

+ ++++ +++

+++

+++

B. Thermolysin (protease/total Rs protein ratio - l/40, 15 min) s3 :;

L7/Ll2 L17 L23

+++ ++++ +

++++ ++++ +++

++++

++++ ++ ++++

+

++

+++ ++++ ++ +++-:

+

*Curlythe proteins which change their accessibility to proteases are shown. The concentration of 9-DMGDMDOT in the mixture is 1 mM; + and -V signs are explained in legend to table I.

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