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L22 Ribosomal Protein and Effect of Its Mutation on Ribosome Resistance to Erythromycin
doi:10.1016/S0022-2836(02)00772-6 available online at http://www.idealibrary.com on
w B
J. Mol. Biol. (2002) 322, 635–644
L22 Ribosomal Protein and Effect of Its Mutation on Ribosome Resistance to Erythromycin Natalia Davydova1*, Victor Streltsov2, Matthew Wilce2, Anders Liljas3 and Maria Garber1 1
Institute of Protein Research Pushchino, Moscow Region 142 290, Russia 2
Crystallography Centre School of Biomedical and Chemical Sciences, University of Western Australia, Crawley 6009, Australia 3
Molecular Biophysics, Lund University, P.O. Box 124 221 00 Lund, Sweden
The ribosomal protein L22 is a core protein of the large ribosomal subunit interacting with all domains of the 23 S rRNA. The triplet Met82-Lys83Arg84 deletion in L22 from Escherichia coli renders cells resistant to erythromycin which is known as an inhibitor of the nascent peptide chain elongation. The crystal structure of the Thermus thermophilus L22 mutant with equivalent triplet Leu82-Lys83-Arg84 deletion has been ˚ resolution. The superpositions of the mutant and determined at 1.8 A the wild-type L22 structures within the 50 S subunits from Haloarcula marismortui and Deinococcus radiodurans show that the mutant b-hairpin is bent inward the ribosome tunnel modifying the shape of its narrowest part and affecting the interaction between L22 and 23 S rRNA. 23 S rRNA nucleotides of domain V participating in erythromycin binding are located on the opposite sides of the tunnel and are brought to those positions by the interaction of the 23 S rRNA with the L22 b-hairpin. The mutation in the L22 b-hairpin affects the orientation and distances between those nucleotides. This destabilizes the erythromycin-binding “pocket” formed by 23 S rRNA nucleotides exposed at the tunnel surface. It seems that erythromycin, while still being able to interact with one side of the tunnel but not reaching the other, is therefore unable to block the polypeptide growth in the drug-resistant ribosome. q 2002 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: erythromycin resistance; crystal structure; mutant; ribosomal protein
Introduction The ribosome, a large ribonucleoprotein complex synthesizing proteins, is a target for a wide variety of antibiotics. While discovery of antibiotics has significantly revolutionized therapeutics of infectious diseases, continuous evolution of bacteria with multiple resistances to drugs substantially reduces their effectiveness. Hence, detailed understanding of antibiotic– ribosome interactions is of particularly important. Activity of most antibiotics is based on their binding to specific areas of the ribosome and affecting the ribosomal function in protein biosynthesis. Macrolide antibiotics, Present address: N. Davydova, Laboratory for Cancer Medicine, Western Australian Institute for Medical Research, Royal Perth Hospital, Perth, WA 6000, Australia. Abbreviation used: wt, wild-type. E-mail address of the corresponding author: [email protected]
including erythromycin, interact with the large ribosomal subunit close to the peptidyl-transferase center and inhibit protein synthesis by impeding the growth of the nascent peptide chain and promoting dissociation of the peptidyl tRNA from the ribosome.1 – 3 The binding site for the best-studied, 14-member-ring macrolide erythromycin was thought to include several positions in domain V of the 23 S rRNA.4 – 7 However, as recently shown,8,9 domain II was also strongly involved in the drug binding. It was noted10 that mutations of selected nucleotides in domain II led to erythromycin resistance and that the 23 S rRNA domains II and V being proximal in the tertiary structure contributed to a single drug-binding pocket. Nevertheless the structural studies11 denied direct influence of domain II (helix 35) as well as any of the ribosomal proteins on the ribosome – erythromycin binding. But, it was admitted there11 that hydrophobic van der Waals or ion-coordinated interactions between the lacton ring and the helix 35 loop of the 23 S rRNA cannot be ruled out and the resistance of
0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved
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Mutant L22 and Ribosome Resistance to Erythromycin
Table 1. Details of data collection and processing Wavelength ˚) Resolution range (A Number of measured reflections Number of unique reflections Completeness (%) Multiplicity Rmerge (%) I/si . 2 R (%) Rfree (%) Rfree test set size ˚ 2) Mean B overall (A ˚ 2) Mean B of solvent atoms (A R.m.s.d. from ideal values ˚) Bond length (A Bond angles (deg.) Dihedral angles (deg.) Improper angles (deg.)
˚ Cu Ka, 1.54179 A 23.2–1.8 64,827 16,162 (1334) 85.6 (47.3) 4 (3) 7.4 (33.1) 84 (57) 20.9 (28.3) 25.5 (28.2) 10 37.8 59.4 0.007 1.27 22.06 0.77
Values given in parentheses are for the highest resolution ˚ ). shell (1.91–1.8 A
macrolides acquired by mutations in L22 and L4 proteins is probably the product of an indirect effect. Although specific antibiotic binding is mainly to the ribosomal rRNA, the ribosomal proteins direct folding and maintain functional three-dimensional structures of the ribosomal RNAs including drugbinding sites. L22, a core protein of the large ribosomal subunit,12,13 interacts with all six domains of the 23 S rRNA.14 It is also the largest contributor to the surface of the tunnel through which the polypeptide product passes,14 and it forms with the L4 protein the narrowest part of this tunnel. The structure of the wild-type Thermus thermophilus L22 has been determined at atomic resolution.13 The L22 molecule consists of a single domain with three a-helices packed against a three-stranded antiparallel b-sheet, which forms a well-packed hydrophobic core. Two of the three strands of the b-structure form an extended b-hairpin which is located between rRNA segments and approximately parallel with the axis of the tunnel in the 50 S subunit.14 Two amino acid residues in the b-hairpin tip along with the neighboring nucleotides from the 23 S RNA domains II and V are responsible for the elongation arrest of the secretion monitor.15 The deletion of three amino acid residues (Escherichia coli Met82-Lys83-Arg84) from the side of the b-hairpin13 renders cells resistant to erythromycin.16 Two of these amino acid residues (Lys83, Arg84) are rather conserved in all known L22 sequences.12,13 Interactions of the ribosomal RNAs and proteins with antibiotics were investigated by different biochemical and genetic techniques (cross-linking, RNA-footprinting, identification of mutations responsible for antibiotic resistance), which pointed to some interactions between a drug and a particular site in the rRNA. X-ray structural studies of the 50 S14,17 and the 30 S18 ribosomal subunits significantly improved the overall picture of intra-ribosome interactions and helped to advance
earlier proposed models for drug– ribosome binding. However, the ribosomal structure itself and even the structures of the small19,20 and large11 subunits co-crystallized with antibiotics as well as electron microscopy study of the erythromycinresistant ribosomes21 cannot fully explain interactions involved. Complementary studies of structural differences between wild-type (wt) and mutated ribosomal elements, which affect antibiotic – ribosome binding, such as L22, should further assist in identifying and understanding interactions responsible for the antibiotic resistance. In the present study, the crystal structure of the Leu82-Lys83-Arg84 (E. coli Met82-Lys83-Arg84) triplet deletion mutant of L22 from T. thermophilus ˚ resolution. Structural changes is reported at 1.8 A of this mutant form of L22 are considered within the whole tertiary structure of the bacterial17 and the archaeal14 large ribosomal subunits.
Results and Discussion Structure description ˚ resolution The structure was determined at 1.8 A through molecular replacement using the coordinates of the T. thermophilus wt L22 (Table 1). The residual electron density maps clarified the structural differences. Two independent monomers A and B in the asymmetric unit of the L22 mutant structure are shown in Figure 1(a). The overall structures of two monomers are very similar, except slight deviations in the flexible region at the N terminus and in the b-hairpin tip. The electron density in the b-hairpin tip of the B molecule was highly diffused between residues Pro87 and Arg99 and was approximately modeled between ˚ ) in the final residues Arg88 and Ile96 (B . 80 A structure. The electron density in the b-hairpin region of the A molecule was reliable and easily traced. The CNS/XtalView 2mFo 2 DFc map (Figure 2) around the b-hairpin tip of the A monomer including the mutation site shows high connectivity along the entire chain and is good as the map calculated for the core chain of the mutant protein. The crystal packing may explain the structural instability in the b-hairpin tip of molecule B. The molecules are packed in the crystal structure in the way that the well-ordered hairpin of ˚ ) mainly with molecule A has contacts (, 4.5 A residues 25– 35 from a-helices of symmetry-related molecules. These helices predominantly consist of highly polar residues, which form stronger contacts with hairpin and keep the rigid packing of the A molecules. On the other hand molecules B face mainly neutral and hydrophobic residues 50 –60, in particular Ala54-Ala55-Ala56, of symmetry-related molecules. This may prevent formation of contacts stabilizing the hairpin of molecule B and it tends to be distorted by the crystal field forces. The following discussion
Mutant L22 and Ribosome Resistance to Erythromycin
637
Figure 1. (a) Structure of a mutant form of the ribosomal protein L22 from T. thermophilus. Two monomers A and B are shown in red and blue, respectively. (b) Superposition of wild-type T. thermophilus (yellow) and H. marismortui (cyan), and mutant T. thermophilus (red) forms. (c) Superposition of wild-type T. thermophilus (yellow) and D. radiodurans (cyan), and mutant T. thermophilus (red) forms.
and Figures will be based on the structure of the well-traced A molecule in the mutant L22 crystal. Wild-type and mutant L22: structural comparison The wild-type Hma and Dra L22 from archaeon Haloarcula marismortui14 (Hma ) and eubacterium
Deinococcus radiodurans17 (Dra ) show high structural homology with its Tth counterpart, except for an additional 30 amino acid RNA-binding loop between a-helices 2 and 3 for Hma, which is characteristic for the archaeal protein, and for an N-terminal 21 amino acid extension for Dra. In fact, the wt Tth L22 structure13 was used as an initial model for the L22 a-chains tracing in the Dra14 and Hma17 50 S subunits.
Figure 2. 2mFo 2 DFc map contoured at 1s around the tip of the b-hairpin from the mutant form of T. thermophilus L22.
638
Mutant L22 and Ribosome Resistance to Erythromycin
Table 2. Correspondence between selected sequences of L22 and 23S for E. coli (Eco ), T. thermophilus (Tth ), D. radiodurans (Dra ) and H. marismortui (Hma ) Eco
Tth
Dra
Hma
Gly79 Pro80 Ala81 Met82 Lys83 Arg84 Ile85 Met86 Pro87 Arg88 Ala89 Lys90 Gly91 Arg92 Ala93 Asp94 Arg95 Ile96 Leu97 Lys98 Arg99
Gly79 Pro80 Ala81 Leu82 Lys83 Arg84 Val85 Leu86 Pro87 Arg88 Ala89 Arg90 Gly91 Arg92 Ala93 Asp94 Ile95 Ile96 Lys97 Lys98 Arg99
Gly100 Pro101 Thr102 Leu103 Lys104 Arg105 Leu106 Ile107 Pro108 Arg109 Ala110 Arg111 Gly112 Ser113 Ala114 Asn115 Ile116 Ile117 Lys118 Lys119 Arg120
Val119 Gly120 Glu121 Gln122 Gln123 Gly124 Arg125 Lys126 Pro127 Arg128 Ala129 Met130 Gly131 Arg132 Ala133 Ser134 Ala135 Trp136 Asn137 Ser138 Pro139
C472 G758 U760 G761 A763 A764 C765 A1275 U1276 G1277 A1278 G1279 U1280 A1630 G1995 A1996 A1997 A1998 U1999 U2000 A2040 A2041 A2042 U2588 U2590 U2592
G467 C838 U840 A841 A843 A844 U845 A1367 U1368 A1369 G1370 A1371 A1372 A1698 G2053 A2054 A2055 C2056 U2057 G2058 C2098 G2099 A2100 C2644 G2646 U2648
L22
Deletion
23 S RNA Domain I Domain II
Domain III IV– V interdomain region
Domain V
C461 G745 U747 G748 A750 A751 A752 U1263 A1264 A1265 G1266 U1267 A1268 A1614 G2012 A2013 A2014 A2015 U2016 U2017 G2057 A2058 A2059 U2609 C2611 U2613
In the present study, the wt Tth and Hma L22 structures13,14 were superimposed using a leastsquares fit (r.m.s. 2.4) with 64 amino acid residues corresponding to the section of the Hma protein after the additional loop to the N terminus including the b-hairpin. The wt Tth and Dra L22 structures13,17 were also superimposed using a least-squares fit (r.m.s. 1.5) with 107 amino acid residues from corresponding sections of Tth 3 –110 aa and Dra 24 –131 aa. Then, the mutant Tth L22 structure was placed over the wt Tth L22 structure in both cases using a least-squares fit (r.m.s. 0.84) with 70 residues from the conserved part of the protein. The superpositions of wt Tth, mutant Tth
and wt Hma L22, and wt Tth, mutant Tth, and wt Dra L22 structures are shown in Figure 1(b) and (c). Without the extra loop in Hma L22 and the extended N terminus in Dra L22, the overall core structures of all proteins are very close. The shape of the b-hairpin in wt Tth, Hma and Dra is also in a good agreement. These similarities justify further structural analysis of the mutant Tth L22 superimposed with wt Dra and Hma L22 within the reported structure of the large ribosomal subunit.14,17 L22 interaction with 23 S RNA The numbering of nucleotide positions and the division of the 23 S RNA molecule into domains is presented according to D. radiodurans sequence.17 Correspondence between E. coli, T. thermophilus, D. radiodurans and H. marismortui numbering is explained in Table 2. Figures 3 and 4 show the superposition of the eubacterial mutant Tth and wt Dra L22 structures in the 50 S subunit from D. radiodurans.17 The superposition within the archaeal H. marismortui 50 S subunit14 gives similar pictures and is not presented here. The structures of the 50 S subunits14,17 show that the b-hairpin of L22 forms a part of the tunnel wall. One side of the b-hairpin loop, which is more exposed to the tunnel surface (Figure 3), experiences the triplet deletion. This triplet has either two polar (Gln in Hma L22) or two charged residues (Lys, Arg in Tth, Dra, Eco L22) with extended side-chains, and one hydrophobic (TthDra Leu/EcoMet/HmaGly) residue (Table 2). All other residues on this side of the b-hairpin are hydrophobic. The opposite side of the b-hairpin has similar set of polar and charged amino acid residues 115 –119, but it faces domain IV of the 23 S rRNA as shown in Figure 3(a). Lys119 is close to, and therefore assumed to be interacting with the phosphate group of G1995. Residues Ile117 and Lys118 interact with A1996, and Ile116 and Asn115 interact with A1997. Pro108 from the mutation side of the loop interacts with A1630 of domain III (Figure 3(b)). The L22 b-hairpin tip (Figure 3(b)) is in close proximity to the nucleotides of domain II (helix 35). In particular, Arg109, Ala110 and Arg111 are close to the phosphate oxygen atoms of G761, A763 and A764, respectively. Gly112 is close to the A764 base. On one side these nucleotides involved in interaction with the b-hairpin tip, but on the other side the A763 base forms a stacking contact with G761 and one H-bond with G758 (Figure 3(b)). The neighboring A764 and C765 bases (Figure 4(a)) protrude inside the tunnel from the same side of the tunnel as the A1630 base (Figure 3). Adjacent U760 forms base-stacking contact with U2592 of domain V and H-bond with A1997 of domain IV. In its turn the U2592 base is H-bonded to the A1996 base (Figure 3(b)). Both A1996 and A1997, while being very close to the L22 b-hairpin residues 115 –118 (Figure 3(a)), form
Mutant L22 and Ribosome Resistance to Erythromycin
639
Figure 3. Tertiary interactions between b-hairpin of L22 and 23 S RNA within the D. radiodurans 50 S subunit: (a) L22 and IV– V interdomain region; (b) L22 and domain II. Short dashed lines indicate some potential hydrogen bonds. The b-hairpin residues of wild-type D. radiodurans (L22Dra ) (cyan) and mutant T. thermophilus (mL22Tth ) (red) L22 forms are shown. Domains backbones of 23 S RNA: II (yellow), III (green), IV– V region (blue), and V (blue) are presented as tubes; selected nucleotides and L22 residues are drawn as licorice and spheres, respectively.
640
Mutant L22 and Ribosome Resistance to Erythromycin
Figure 4. (a) The tertiary structure of the D. radiodurans 23 S RNA with erythromycin-binding site. The b-hairpins of wild-type D. radiodurans (L22Dra ) (cyan) and mutant T. thermophilus (mL22Tth ) (red), and erythromycin molecule (cyan with O red) are shown. Short dashed lines indicate potential hydrogen bonds. (b) The tertiary structure of the 23 S RNA, wildtype (cyan) and mutant (red) forms of L22, and L4 (black) in proximity to the narrowest part of the polypeptide exit tunnel. Short dashed lines indicate distances between selected residues and nucleotides. Domains backbones of 23 S RNA are color-coded: I (purple), II (yellow), III (green), IV– V region (blue), and V (blue). Selected nucleotides and L22 residues are indicated as in Figure 3.
Mutant L22 and Ribosome Resistance to Erythromycin
H-bonds with G1279 and G1277 of the II –III interdomain region, respectively (not shown). The observed interactions demonstrate that L22 plays role of a fastener for holding domains II – V of 23 S RNA together and directs their proper folding and relative positions in the large subunit assembly. This is in agreement with the earlier chemical modification studies22 and is supported by the fact that any small mutation in this protein strongly affects ribosomal assembly and function.23 – 25 Effect of L22 mutation on the ribosome– erythromycin binding The reported26 structures of the erythromycin A and B molecules are very similar except that erythromycin A has an extra hydroxyl group at the position 12 of the lactone ring. Distances from the active hydroxyl oxygen attached to the cladinose to that at position 11 in both A and B structures and to that at position 12 of the lactone ˚ and 12.3 A ˚ , respectively. ring in A are , 10.6 A However, the erythromycin A structure from the D. radiodurans ribosome –erythromycin A complex11 has significantly different conformation ˚ resolution electron within the accuracy of the 3.5 A density measured. The above-mentioned distances ˚ and in this erythromycin A structure are 5.4 A ˚ 7.8 A, respectively. Such erythromycin molecule ˚ (considering sidepenetrated through the 13 A chains) entry opening near the peptidyl-transferase ˚ center into the tunnel down to the narrow 9.8 A part between the U2588 and A2045 nucleotides ˚ length H-bonds with them forming less than 4 A and with A2041, A2042 and G2484 as reported11 and shown in Figure 4. This agrees with the earlier observation27 that A2041Dra/G2099Hma/A2058Eco is the key drug contact point within domain V. However, results11 does not support the footprinting studies,8 – 10 which showed the involvement of domain II (helix 35) in the erythromycin – ribosome binding. Our structural study helps to clarify the discrepancy between those observations. Enhanced accessibility of the N1 position of A752Eco/ C765Dra/U845Hma in the erythromycin-bound ribosome8,9 could be due to interaction of the lactone ring with U2588Dra (Ery(O13) – ˚ ), which in turn interacts with U2588(N3) ¼ 3.4 A ˚ ) (Figure C765Dra (U2588(O2) – C765(N4) ¼ 3.3 A 4(a)). According to the observed binding11 erythromycin blocks partially the ribosome tunnel, but does not interfere directly with the L22 b-loop as shown in Figure 4(a). However, L22 with its elongated b-hairpin plays an important role in bringing together domains II – V of 23 S rRNA. These domains form strong contacts between each other and expose nucleotides of domain V into the tunnel. The erythromycin-bound U2588 interacts with C765 as well as with G758 (U2588(O2) – ˚ ) as shown in Figure 4(a). G758 is G758(O6) ¼ 4 A also in close contacts with C765 (C765(N3)–
641 ˚ ) and A763 (A763(N1)–G758(N2)¼ G758(N1) ¼ 2.9 A ˚ ), (A763(N6) – G758(N3) ¼ 2.9 A ˚ ), (A763(N6) – 2.8 A ˚ ˚ ). G758(O2) ¼ 3.0 A), (A763(N6) –G758(N2) ¼ 3.8 A The A763 base forms a stacking contact with ˚ and G761 interG761 at the distances about 3.3 A acts with domain V via C2591 (G761(O1)– ˚ ). This indicates that domains V C2591(N4) ¼ 2.9 A and II are in close contact and form an interaction chain through U2588, C765, G758, A764, A763 and G761 nucleotides. The one end of this chain (U2588) interacts with erythromycin while the other one (A764, A763 and G761) is in close contact with the L22 b-hairpin (Arg109-Gly112) (Figure 3(b)). As mentioned above, the other points of erythromycin interaction are G2484, A2045, A2042 and A2041 of domain V (Figure 4(a)). But A2041 inter˚ ). acts with U2000 (A2040(O1) – U2000(O2) ¼ 3.7 A The neighboring A2040 has contact with U1999 ˚ ) of IV– V inter(U1999(O3) –A2040(O3) ¼ 3.0 A domain region (Figure 4(a)), and this part of interdomain region, particularly A1997, A1996 and G1995, has strong interaction with less distorted part of the L22 b-hairpin (Figure 3(a)). Thus the L22 b-hairpin triplet deletion results in significant bending of the b-hairpin inside the tunnel that modifies the shape of its narrowest part formed by L4 and L22 (Figure 4(b)). The initial ˚ between a-chains of L4 and opening of about 12 A ˚ (side-chains are not L22 is reduced to 8.5– 9 A considered) due to bending of the b-hairpin tip. However, it would not inhibit the growth of the nascent polypeptide, because the polypeptide ˚ in diameter, and it seems also that it chain is , 6 A would not protect the ribosome from erythromycin binding either. It is known4 that erythromycin can bind to the resistant ribosomes without inhibiting the protein synthesis. Recent cryo-electron microscopy studies21 of the E. coli 70 S erythromycin-resistant ribosome with the L22 triplet deletion mutant help to explain that fact. It was demonstrated that the tunnel entry opening in the ribosome with the L22 mutant was twice that opening in the wt ribosome and was comparable with the opening in the initiator tRNA-complexed ribosome. It should be noted that the study21 also revealed the formation of an additional cavity between the beginning of the tunnel and the tip of protein L22, which would render the binding of erythromycin to the ribosome with the mutant L22 ineffective. It seems that this cavity is the space formed between the bent b-hairpin tip and the distorted 23 S rRNA domains II and V in Figure 4(a) and (b). This entirely agrees with the suggestion21 that the cavity in immediate vicinity to the L22 tip “might be formed as a result of mutation in the tip” and “it possibly involves reorganization in the central loop of domain V, induced by a transition in helix 35 of domain II”. It can be concluded now that the L22 triplet deletion mutation may lead to the following changes: it makes the side of the b-hairpin that is exposed to the tunnel surface highly hydrophobic
642
by removing polar or charged and strongly basic residues from the loop; it changes the overall orientation of the tip of the hairpin and disrupts its binding via Arg109-Gly112 to nucleotides G761, A763 and A764 of the 23 S rRNA domain II, which are in strong contact with U2588 of domain V—a binding point of erythromycin as reported;11 it bends the hairpin inwards the narrow part of the tunnel. A similar triplet deletion on the other side of the b-hairpin would be unfavorable. It may also put at risk the stability of whole assembly due to stronger interactions of that side with 23 S rRNA. The bending of b-hairpin weakens the contact between L22 and domain IV, however, residues 116– 119 in mutant L22 should still be very close to A1996-G1995 and the second interacting pocket of erythromycin via domain V (2041, 2042, 2045 and 2484), which is in contact with this part of domain IV, is less disturbed. Taking into account ˚ resolution) and conan approximate (at 3.5 A siderably different conformation of the 50 S bound erythromycin11 compare to its free structure26 and the dynamic behavior of the tunnel opening depending on the ribosome function21 we may speculate that there is also a possibility for the direct interaction between erythromycin and the A764 or C765 nucleotides exposed to the tunnel (Figure 4(a)). The whole undistorted erythromycin ˚ space molecule26 can be fitted well into , 15 A between the A764 and A2041 nucleotides. The nucleotides participating in erythromycin binding are located on opposite sides of the tunnel and are brought to those positions by interactions of domains II –V with L22. If there is mechanical blocking of polypeptide elongation by erythromycin as suggested by cryo-microscopy study,21 then it can be assumed that erythromycin, bound to the opposite walls of the tunnel, works as a gate interfering with further growth and progress of polypeptide after formation of a chain of 2 – 5 amino acid residues. Mutations in 23 S rRNA and/or in the L22 b-hairpin affect the orientation and distances between nucleotides, which participate in the binding of this antibiotic. Erythromycin, while still being able to bind to one of the sides of the tunnel, is unable to block the polypeptide growth in the drug-resistant ribosome.
Materials and Methods Protein expression, purification and crystallization The mutant protein was overexpressed, purified and crystallized as described earlier.12,28 Single plate-like 1.0 mm £ 0.5 mm £ 0.13 mm crystals have been obtained by the hanging drop vapor-diffusion technique. The drops were set up by mixing 2 ml of precipitant solution on siliconized cover slides and equilibrated against 1.0 ml of the same precipitant solution. Crystallization trays were incubated at 20 8C. The space group is P21, b ¼ 86:02 A; c¼ with cell dimensions a ¼ 31:79 A; b ¼ 104:38: 38:56 A;
Mutant L22 and Ribosome Resistance to Erythromycin
Data collection Selected crystals were mounted in thin-walled quartz capillaries, which were sealed with wax after filling both ends with reservoir solution. X-ray diffraction data were collected at room temperature using an MAR 345 Image Plate area detector (MarResearch, Hamburg, Germany) using X-rays from a Rigaku RU-200 rotating anode generator operating at 40 kV and 100 mA. X-rays were focused using nickel-coated mirrors. Due to crystal deterioration only 203 frames of 0.58 Dw oscillation were collected. However, it gave the completeness of 86%, which was enough to refine the structure to acceptable R-factors. The lack of actually observed data due to low intensity statistics is most noticeable in higher resolution shells (Table 1). Attempted low temperature data collection was less successful due to problems with even more rapid deterioration of protein crystals. Indexing, integration and scaling of data sets were carried out using DENZO and SCALEPACK.29 Structure determination The crystal structure was solved by the molecular replacement method using the CNS package.30 As a search model, a truncated version of water-free wt L22 (PDB accession number 1BXE13) structure was generated by removing the non-conserved b-hairpin region. The initial rotation search suggested that two molecules were in the asymmetric unit. The translation search was then used to determine which of the rotation solutions correspond to the two monomers. The final correlation coefficient of 0.577 and the packing value of 0.63 were significantly greater than those for the translation search with only one molecule. The positions of the two molecules A and B in the asymmetric unit were optimized to R ¼ 0.39 with the CNS30 rigid body leastsquares refinement using the whole available resolution range. Rfree cross-validation index based on randomly selected reflections (10% of the total set) was employed to monitor the subsequent refinement of the model. Following rigid body refinement, simulated annealing using torsion angle dynamics was used to improve the model. After that, b-hairpins for the two independent molecules were built into the generated density maps. Both sa-weighted 2mFo 2 DFc and mFo 2 DFc density maps30 were calculated and examined using the O program31 and XtalView.32 The electron density in the loop region of one molecule A was easily traced, while the electron density in the loop tip of the molecule B was highly diffused (residues 88 – 99), possibly as a result of molecule-packing difference. However, the latter was approximately traced and included in the model with ˚ 2). There was also a poor very high B-factors (70 – 116 A density in the C-terminal regions (residues 110 – 113) of both molecules. The side-chains were further adjusted stepwise during the course of refinement using restrained individual B-factor refinement followed by energy minimization of the coordinates. During the final stages of refinement, water molecules were inserted into the model only if there were peaks in the 2mFo 2 DFc maps higher than 2.0s with proper hydrogen-bonding distances. Water molecules with temperature factors ˚ 2 and real space correlation less than greater than 80 A 0.5 were excluded from subsequent steps of refinement. The final model containing 1738 protein non-H atoms, 37 solvent molecules gives an R factor of 20.9% (Rfree ¼ 25.5%). Assessment of the quality of the final structure showed that 91.4% of the residues falling in
643
Mutant L22 and Ribosome Resistance to Erythromycin
the most-favored region and 1% is in disallowed region of the PROCHECK Ramachandran plot.33 The latter residues are from the b-hairpin tip of the molecule B with poorly defined density. Further details of data collection, refinement statistics and final model quality are given in Table 1. All the Figures were generated using the program VMD.34 Accession number The coordinates for the mutant L22 have been deposited in the Brookhaven Protein Data Bank. The accession number is 1I4J.
Acknowledgments This work was supported by the Australian Research Council, the Russian Academy of Sciences and the Russian Foundation for Basic research. The research of M.B.G. was supported in part by the International Research Scholar’s award from the Howard Hughes Medical Institute.
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23 S rRNA reveals rRNA folding required for formation of macrolide-binding site. Mol. Microbiol. 31, 633–639. Douthwaite, S., Hansen, L. H. & Mauvais, P. (2000). Macrolide– ketolide inhibition of MLS-resistant ribosomes is improved by alternative drug interaction with domain II of 23 S rRNA. Mol. Microbiol. 36, 183 –193. Schlunzen, F., Zarivach, R., Harms, J., Bashan, A., Tocilj, A., Albrecht, R. et al. (2001). Structural basis for the interaction of antibiotics with the peptidyl transferase center in eubacteria. Nature, 413, 814– 821. Davydova, N. L., Gryaznova, O. I., Mashchenko, O. V., Vysotskaya, V. S., Jonsson, B.-H., Al-Karadaghi, S. et al. (1995). Ribosomal protein L22 from Thermus thermophilus: sequencing, overexpression and crystallisation. FEBS Letters, 369, 229– 232. ˚ berg, A., Al-Kharadaghi, S., Nikulin, A., Unge, J., A Nikonov, S., Davydova, N. et al. (1998). The crystal structure of ribosomal protein L22 from Thermus thermophilus: insights into the mechanism of erythromycin resistance. Structure, 6, 1577– 1586. Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000). The complete atomic structure of the ˚ resolution. Science, large ribosomal subunit at 2.4 A 289, 905– 920. Nakatogawa, H. & Ito, K. (2002). The ribosomal exit tunnel functions as a discriminating gate. Cell, 108, 629 –636. Chittum, H. S. & Champney, W. S. (1994). Ribosomal protein gene sequence changes in erythromycinresistant mutants of Escherichia coli. J. Bacteriol. 176, 6192 –6198. Harms, J., Schluenzen, F., Zarivach, R., Bashan, A., Gat, S., Agmon, I. et al. (2001). High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell, 107, 679 –688. Wimberly, B. T., Broadersen, D. E., Clemons, W. M., Jr, Morgan-Warren, R. J., Carter, A. P., Vonrhein, C. et al. (2000). Structure of the 30S ribosomal subunit. Nature, 407, 327– 339. Carter, A. P., Clemons, W. M., Jr, Brodersen, D. E., Morgan-Warren, R. J., Wimberly, B. T. & Ramakrishnan, V. (2000). Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature, 407, 340– 348. Brodersen, D. E., Clemons, W. M., Jr, Carter, A. P., Morgan-Warren, R. J., Wimberly, B. T. & Ramakrishnan, V. (2000). The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell, 103, 1143– 1154. Gabashvili, I. S., Gregory, S. T., Valle, M., Grassucci, R., Worbs, M., Wahl, M. C. et al. (2001). The polypeptide tunnel system in the ribosome and its gating in erythromycin resistance mutants of L4 and L22. Mol. Cell, 8, 181– 188. Gregory, S. T. & Dahlberg, A. E. (1999). Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA. J. Mol. Biol. 289, 827– 834. Champney, W. S. (1989). Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins. Biochim. Biophys. Acta, 609, 464– 474. Burnette-Vick, B., Champney, W. S. & Musich, P. R. (1994). A temperature sensitive mutant of Escherichia coli with an alteration in ribosomal protein L22. Genetica, 94, 17 –25.
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25. Chittum, H. S. & Champney, W. S. (1995). Erythromycin inhibits the assembly of the large ribosomal subunit in growing Escherichia coli cells. Curr. Microbiol. 30, 273– 279. 26. Stephenson, G. A., Stowell, J. G., Toma, P. H., Pfeiffer, R. R. & Byrn, S. R. (1997). Solid-state investigations of erythromycin A dihydrate: structure, NMR spectroscopy, and hygroscopicity. J. Pharm. Sci. 86, 1239– 1244. 27. Moazed, D. & Noller, H. F. (1987). Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping sites in the peptidyl transferase region of 23 S ribosomal RNA. Biochimie, 69, 879– 884. 28. Davydova, N., Fedorov, R., Streltsov, V., Liljas, A. & Garber, M. (2001). Crystals of a mutant form of ribosomal protein L22 rendering bacterial ribosomes resistant to erythromycin. Acta Crystallog. sect. D, 57, 1150 –1152. 29. Otwinowski, Z. & Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307– 326.
30. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998). Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallog. sect. D, 54, 905– 921. 31. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallog. sect. A, 47, 110 – 119. 32. McRee, D. E. (1999). XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156– 165. 33. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallog. 26, 283– 291. 34. Humphrey, W., Dalke, A. & Shulten, K. (1996). VMD—visual molecular dynamics. J. Mol. Graph. 14.1, 33 – 38.
Edited by J. Doudna (Received 9 May 2002; received in revised form 15 July 2002; accepted 22 July 2002)
w B
J. Mol. Biol. (2002) 322, 635–644
L22 Ribosomal Protein and Effect of Its Mutation on Ribosome Resistance to Erythromycin Natalia Davydova1*, Victor Streltsov2, Matthew Wilce2, Anders Liljas3 and Maria Garber1 1
Institute of Protein Research Pushchino, Moscow Region 142 290, Russia 2
Crystallography Centre School of Biomedical and Chemical Sciences, University of Western Australia, Crawley 6009, Australia 3
Molecular Biophysics, Lund University, P.O. Box 124 221 00 Lund, Sweden
The ribosomal protein L22 is a core protein of the large ribosomal subunit interacting with all domains of the 23 S rRNA. The triplet Met82-Lys83Arg84 deletion in L22 from Escherichia coli renders cells resistant to erythromycin which is known as an inhibitor of the nascent peptide chain elongation. The crystal structure of the Thermus thermophilus L22 mutant with equivalent triplet Leu82-Lys83-Arg84 deletion has been ˚ resolution. The superpositions of the mutant and determined at 1.8 A the wild-type L22 structures within the 50 S subunits from Haloarcula marismortui and Deinococcus radiodurans show that the mutant b-hairpin is bent inward the ribosome tunnel modifying the shape of its narrowest part and affecting the interaction between L22 and 23 S rRNA. 23 S rRNA nucleotides of domain V participating in erythromycin binding are located on the opposite sides of the tunnel and are brought to those positions by the interaction of the 23 S rRNA with the L22 b-hairpin. The mutation in the L22 b-hairpin affects the orientation and distances between those nucleotides. This destabilizes the erythromycin-binding “pocket” formed by 23 S rRNA nucleotides exposed at the tunnel surface. It seems that erythromycin, while still being able to interact with one side of the tunnel but not reaching the other, is therefore unable to block the polypeptide growth in the drug-resistant ribosome. q 2002 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: erythromycin resistance; crystal structure; mutant; ribosomal protein
Introduction The ribosome, a large ribonucleoprotein complex synthesizing proteins, is a target for a wide variety of antibiotics. While discovery of antibiotics has significantly revolutionized therapeutics of infectious diseases, continuous evolution of bacteria with multiple resistances to drugs substantially reduces their effectiveness. Hence, detailed understanding of antibiotic– ribosome interactions is of particularly important. Activity of most antibiotics is based on their binding to specific areas of the ribosome and affecting the ribosomal function in protein biosynthesis. Macrolide antibiotics, Present address: N. Davydova, Laboratory for Cancer Medicine, Western Australian Institute for Medical Research, Royal Perth Hospital, Perth, WA 6000, Australia. Abbreviation used: wt, wild-type. E-mail address of the corresponding author: [email protected]
including erythromycin, interact with the large ribosomal subunit close to the peptidyl-transferase center and inhibit protein synthesis by impeding the growth of the nascent peptide chain and promoting dissociation of the peptidyl tRNA from the ribosome.1 – 3 The binding site for the best-studied, 14-member-ring macrolide erythromycin was thought to include several positions in domain V of the 23 S rRNA.4 – 7 However, as recently shown,8,9 domain II was also strongly involved in the drug binding. It was noted10 that mutations of selected nucleotides in domain II led to erythromycin resistance and that the 23 S rRNA domains II and V being proximal in the tertiary structure contributed to a single drug-binding pocket. Nevertheless the structural studies11 denied direct influence of domain II (helix 35) as well as any of the ribosomal proteins on the ribosome – erythromycin binding. But, it was admitted there11 that hydrophobic van der Waals or ion-coordinated interactions between the lacton ring and the helix 35 loop of the 23 S rRNA cannot be ruled out and the resistance of
0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved
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Mutant L22 and Ribosome Resistance to Erythromycin
Table 1. Details of data collection and processing Wavelength ˚) Resolution range (A Number of measured reflections Number of unique reflections Completeness (%) Multiplicity Rmerge (%) I/si . 2 R (%) Rfree (%) Rfree test set size ˚ 2) Mean B overall (A ˚ 2) Mean B of solvent atoms (A R.m.s.d. from ideal values ˚) Bond length (A Bond angles (deg.) Dihedral angles (deg.) Improper angles (deg.)
˚ Cu Ka, 1.54179 A 23.2–1.8 64,827 16,162 (1334) 85.6 (47.3) 4 (3) 7.4 (33.1) 84 (57) 20.9 (28.3) 25.5 (28.2) 10 37.8 59.4 0.007 1.27 22.06 0.77
Values given in parentheses are for the highest resolution ˚ ). shell (1.91–1.8 A
macrolides acquired by mutations in L22 and L4 proteins is probably the product of an indirect effect. Although specific antibiotic binding is mainly to the ribosomal rRNA, the ribosomal proteins direct folding and maintain functional three-dimensional structures of the ribosomal RNAs including drugbinding sites. L22, a core protein of the large ribosomal subunit,12,13 interacts with all six domains of the 23 S rRNA.14 It is also the largest contributor to the surface of the tunnel through which the polypeptide product passes,14 and it forms with the L4 protein the narrowest part of this tunnel. The structure of the wild-type Thermus thermophilus L22 has been determined at atomic resolution.13 The L22 molecule consists of a single domain with three a-helices packed against a three-stranded antiparallel b-sheet, which forms a well-packed hydrophobic core. Two of the three strands of the b-structure form an extended b-hairpin which is located between rRNA segments and approximately parallel with the axis of the tunnel in the 50 S subunit.14 Two amino acid residues in the b-hairpin tip along with the neighboring nucleotides from the 23 S RNA domains II and V are responsible for the elongation arrest of the secretion monitor.15 The deletion of three amino acid residues (Escherichia coli Met82-Lys83-Arg84) from the side of the b-hairpin13 renders cells resistant to erythromycin.16 Two of these amino acid residues (Lys83, Arg84) are rather conserved in all known L22 sequences.12,13 Interactions of the ribosomal RNAs and proteins with antibiotics were investigated by different biochemical and genetic techniques (cross-linking, RNA-footprinting, identification of mutations responsible for antibiotic resistance), which pointed to some interactions between a drug and a particular site in the rRNA. X-ray structural studies of the 50 S14,17 and the 30 S18 ribosomal subunits significantly improved the overall picture of intra-ribosome interactions and helped to advance
earlier proposed models for drug– ribosome binding. However, the ribosomal structure itself and even the structures of the small19,20 and large11 subunits co-crystallized with antibiotics as well as electron microscopy study of the erythromycinresistant ribosomes21 cannot fully explain interactions involved. Complementary studies of structural differences between wild-type (wt) and mutated ribosomal elements, which affect antibiotic – ribosome binding, such as L22, should further assist in identifying and understanding interactions responsible for the antibiotic resistance. In the present study, the crystal structure of the Leu82-Lys83-Arg84 (E. coli Met82-Lys83-Arg84) triplet deletion mutant of L22 from T. thermophilus ˚ resolution. Structural changes is reported at 1.8 A of this mutant form of L22 are considered within the whole tertiary structure of the bacterial17 and the archaeal14 large ribosomal subunits.
Results and Discussion Structure description ˚ resolution The structure was determined at 1.8 A through molecular replacement using the coordinates of the T. thermophilus wt L22 (Table 1). The residual electron density maps clarified the structural differences. Two independent monomers A and B in the asymmetric unit of the L22 mutant structure are shown in Figure 1(a). The overall structures of two monomers are very similar, except slight deviations in the flexible region at the N terminus and in the b-hairpin tip. The electron density in the b-hairpin tip of the B molecule was highly diffused between residues Pro87 and Arg99 and was approximately modeled between ˚ ) in the final residues Arg88 and Ile96 (B . 80 A structure. The electron density in the b-hairpin region of the A molecule was reliable and easily traced. The CNS/XtalView 2mFo 2 DFc map (Figure 2) around the b-hairpin tip of the A monomer including the mutation site shows high connectivity along the entire chain and is good as the map calculated for the core chain of the mutant protein. The crystal packing may explain the structural instability in the b-hairpin tip of molecule B. The molecules are packed in the crystal structure in the way that the well-ordered hairpin of ˚ ) mainly with molecule A has contacts (, 4.5 A residues 25– 35 from a-helices of symmetry-related molecules. These helices predominantly consist of highly polar residues, which form stronger contacts with hairpin and keep the rigid packing of the A molecules. On the other hand molecules B face mainly neutral and hydrophobic residues 50 –60, in particular Ala54-Ala55-Ala56, of symmetry-related molecules. This may prevent formation of contacts stabilizing the hairpin of molecule B and it tends to be distorted by the crystal field forces. The following discussion
Mutant L22 and Ribosome Resistance to Erythromycin
637
Figure 1. (a) Structure of a mutant form of the ribosomal protein L22 from T. thermophilus. Two monomers A and B are shown in red and blue, respectively. (b) Superposition of wild-type T. thermophilus (yellow) and H. marismortui (cyan), and mutant T. thermophilus (red) forms. (c) Superposition of wild-type T. thermophilus (yellow) and D. radiodurans (cyan), and mutant T. thermophilus (red) forms.
and Figures will be based on the structure of the well-traced A molecule in the mutant L22 crystal. Wild-type and mutant L22: structural comparison The wild-type Hma and Dra L22 from archaeon Haloarcula marismortui14 (Hma ) and eubacterium
Deinococcus radiodurans17 (Dra ) show high structural homology with its Tth counterpart, except for an additional 30 amino acid RNA-binding loop between a-helices 2 and 3 for Hma, which is characteristic for the archaeal protein, and for an N-terminal 21 amino acid extension for Dra. In fact, the wt Tth L22 structure13 was used as an initial model for the L22 a-chains tracing in the Dra14 and Hma17 50 S subunits.
Figure 2. 2mFo 2 DFc map contoured at 1s around the tip of the b-hairpin from the mutant form of T. thermophilus L22.
638
Mutant L22 and Ribosome Resistance to Erythromycin
Table 2. Correspondence between selected sequences of L22 and 23S for E. coli (Eco ), T. thermophilus (Tth ), D. radiodurans (Dra ) and H. marismortui (Hma ) Eco
Tth
Dra
Hma
Gly79 Pro80 Ala81 Met82 Lys83 Arg84 Ile85 Met86 Pro87 Arg88 Ala89 Lys90 Gly91 Arg92 Ala93 Asp94 Arg95 Ile96 Leu97 Lys98 Arg99
Gly79 Pro80 Ala81 Leu82 Lys83 Arg84 Val85 Leu86 Pro87 Arg88 Ala89 Arg90 Gly91 Arg92 Ala93 Asp94 Ile95 Ile96 Lys97 Lys98 Arg99
Gly100 Pro101 Thr102 Leu103 Lys104 Arg105 Leu106 Ile107 Pro108 Arg109 Ala110 Arg111 Gly112 Ser113 Ala114 Asn115 Ile116 Ile117 Lys118 Lys119 Arg120
Val119 Gly120 Glu121 Gln122 Gln123 Gly124 Arg125 Lys126 Pro127 Arg128 Ala129 Met130 Gly131 Arg132 Ala133 Ser134 Ala135 Trp136 Asn137 Ser138 Pro139
C472 G758 U760 G761 A763 A764 C765 A1275 U1276 G1277 A1278 G1279 U1280 A1630 G1995 A1996 A1997 A1998 U1999 U2000 A2040 A2041 A2042 U2588 U2590 U2592
G467 C838 U840 A841 A843 A844 U845 A1367 U1368 A1369 G1370 A1371 A1372 A1698 G2053 A2054 A2055 C2056 U2057 G2058 C2098 G2099 A2100 C2644 G2646 U2648
L22
Deletion
23 S RNA Domain I Domain II
Domain III IV– V interdomain region
Domain V
C461 G745 U747 G748 A750 A751 A752 U1263 A1264 A1265 G1266 U1267 A1268 A1614 G2012 A2013 A2014 A2015 U2016 U2017 G2057 A2058 A2059 U2609 C2611 U2613
In the present study, the wt Tth and Hma L22 structures13,14 were superimposed using a leastsquares fit (r.m.s. 2.4) with 64 amino acid residues corresponding to the section of the Hma protein after the additional loop to the N terminus including the b-hairpin. The wt Tth and Dra L22 structures13,17 were also superimposed using a least-squares fit (r.m.s. 1.5) with 107 amino acid residues from corresponding sections of Tth 3 –110 aa and Dra 24 –131 aa. Then, the mutant Tth L22 structure was placed over the wt Tth L22 structure in both cases using a least-squares fit (r.m.s. 0.84) with 70 residues from the conserved part of the protein. The superpositions of wt Tth, mutant Tth
and wt Hma L22, and wt Tth, mutant Tth, and wt Dra L22 structures are shown in Figure 1(b) and (c). Without the extra loop in Hma L22 and the extended N terminus in Dra L22, the overall core structures of all proteins are very close. The shape of the b-hairpin in wt Tth, Hma and Dra is also in a good agreement. These similarities justify further structural analysis of the mutant Tth L22 superimposed with wt Dra and Hma L22 within the reported structure of the large ribosomal subunit.14,17 L22 interaction with 23 S RNA The numbering of nucleotide positions and the division of the 23 S RNA molecule into domains is presented according to D. radiodurans sequence.17 Correspondence between E. coli, T. thermophilus, D. radiodurans and H. marismortui numbering is explained in Table 2. Figures 3 and 4 show the superposition of the eubacterial mutant Tth and wt Dra L22 structures in the 50 S subunit from D. radiodurans.17 The superposition within the archaeal H. marismortui 50 S subunit14 gives similar pictures and is not presented here. The structures of the 50 S subunits14,17 show that the b-hairpin of L22 forms a part of the tunnel wall. One side of the b-hairpin loop, which is more exposed to the tunnel surface (Figure 3), experiences the triplet deletion. This triplet has either two polar (Gln in Hma L22) or two charged residues (Lys, Arg in Tth, Dra, Eco L22) with extended side-chains, and one hydrophobic (TthDra Leu/EcoMet/HmaGly) residue (Table 2). All other residues on this side of the b-hairpin are hydrophobic. The opposite side of the b-hairpin has similar set of polar and charged amino acid residues 115 –119, but it faces domain IV of the 23 S rRNA as shown in Figure 3(a). Lys119 is close to, and therefore assumed to be interacting with the phosphate group of G1995. Residues Ile117 and Lys118 interact with A1996, and Ile116 and Asn115 interact with A1997. Pro108 from the mutation side of the loop interacts with A1630 of domain III (Figure 3(b)). The L22 b-hairpin tip (Figure 3(b)) is in close proximity to the nucleotides of domain II (helix 35). In particular, Arg109, Ala110 and Arg111 are close to the phosphate oxygen atoms of G761, A763 and A764, respectively. Gly112 is close to the A764 base. On one side these nucleotides involved in interaction with the b-hairpin tip, but on the other side the A763 base forms a stacking contact with G761 and one H-bond with G758 (Figure 3(b)). The neighboring A764 and C765 bases (Figure 4(a)) protrude inside the tunnel from the same side of the tunnel as the A1630 base (Figure 3). Adjacent U760 forms base-stacking contact with U2592 of domain V and H-bond with A1997 of domain IV. In its turn the U2592 base is H-bonded to the A1996 base (Figure 3(b)). Both A1996 and A1997, while being very close to the L22 b-hairpin residues 115 –118 (Figure 3(a)), form
Mutant L22 and Ribosome Resistance to Erythromycin
639
Figure 3. Tertiary interactions between b-hairpin of L22 and 23 S RNA within the D. radiodurans 50 S subunit: (a) L22 and IV– V interdomain region; (b) L22 and domain II. Short dashed lines indicate some potential hydrogen bonds. The b-hairpin residues of wild-type D. radiodurans (L22Dra ) (cyan) and mutant T. thermophilus (mL22Tth ) (red) L22 forms are shown. Domains backbones of 23 S RNA: II (yellow), III (green), IV– V region (blue), and V (blue) are presented as tubes; selected nucleotides and L22 residues are drawn as licorice and spheres, respectively.
640
Mutant L22 and Ribosome Resistance to Erythromycin
Figure 4. (a) The tertiary structure of the D. radiodurans 23 S RNA with erythromycin-binding site. The b-hairpins of wild-type D. radiodurans (L22Dra ) (cyan) and mutant T. thermophilus (mL22Tth ) (red), and erythromycin molecule (cyan with O red) are shown. Short dashed lines indicate potential hydrogen bonds. (b) The tertiary structure of the 23 S RNA, wildtype (cyan) and mutant (red) forms of L22, and L4 (black) in proximity to the narrowest part of the polypeptide exit tunnel. Short dashed lines indicate distances between selected residues and nucleotides. Domains backbones of 23 S RNA are color-coded: I (purple), II (yellow), III (green), IV– V region (blue), and V (blue). Selected nucleotides and L22 residues are indicated as in Figure 3.
Mutant L22 and Ribosome Resistance to Erythromycin
H-bonds with G1279 and G1277 of the II –III interdomain region, respectively (not shown). The observed interactions demonstrate that L22 plays role of a fastener for holding domains II – V of 23 S RNA together and directs their proper folding and relative positions in the large subunit assembly. This is in agreement with the earlier chemical modification studies22 and is supported by the fact that any small mutation in this protein strongly affects ribosomal assembly and function.23 – 25 Effect of L22 mutation on the ribosome– erythromycin binding The reported26 structures of the erythromycin A and B molecules are very similar except that erythromycin A has an extra hydroxyl group at the position 12 of the lactone ring. Distances from the active hydroxyl oxygen attached to the cladinose to that at position 11 in both A and B structures and to that at position 12 of the lactone ˚ and 12.3 A ˚ , respectively. ring in A are , 10.6 A However, the erythromycin A structure from the D. radiodurans ribosome –erythromycin A complex11 has significantly different conformation ˚ resolution electron within the accuracy of the 3.5 A density measured. The above-mentioned distances ˚ and in this erythromycin A structure are 5.4 A ˚ 7.8 A, respectively. Such erythromycin molecule ˚ (considering sidepenetrated through the 13 A chains) entry opening near the peptidyl-transferase ˚ center into the tunnel down to the narrow 9.8 A part between the U2588 and A2045 nucleotides ˚ length H-bonds with them forming less than 4 A and with A2041, A2042 and G2484 as reported11 and shown in Figure 4. This agrees with the earlier observation27 that A2041Dra/G2099Hma/A2058Eco is the key drug contact point within domain V. However, results11 does not support the footprinting studies,8 – 10 which showed the involvement of domain II (helix 35) in the erythromycin – ribosome binding. Our structural study helps to clarify the discrepancy between those observations. Enhanced accessibility of the N1 position of A752Eco/ C765Dra/U845Hma in the erythromycin-bound ribosome8,9 could be due to interaction of the lactone ring with U2588Dra (Ery(O13) – ˚ ), which in turn interacts with U2588(N3) ¼ 3.4 A ˚ ) (Figure C765Dra (U2588(O2) – C765(N4) ¼ 3.3 A 4(a)). According to the observed binding11 erythromycin blocks partially the ribosome tunnel, but does not interfere directly with the L22 b-loop as shown in Figure 4(a). However, L22 with its elongated b-hairpin plays an important role in bringing together domains II – V of 23 S rRNA. These domains form strong contacts between each other and expose nucleotides of domain V into the tunnel. The erythromycin-bound U2588 interacts with C765 as well as with G758 (U2588(O2) – ˚ ) as shown in Figure 4(a). G758 is G758(O6) ¼ 4 A also in close contacts with C765 (C765(N3)–
641 ˚ ) and A763 (A763(N1)–G758(N2)¼ G758(N1) ¼ 2.9 A ˚ ), (A763(N6) – G758(N3) ¼ 2.9 A ˚ ), (A763(N6) – 2.8 A ˚ ˚ ). G758(O2) ¼ 3.0 A), (A763(N6) –G758(N2) ¼ 3.8 A The A763 base forms a stacking contact with ˚ and G761 interG761 at the distances about 3.3 A acts with domain V via C2591 (G761(O1)– ˚ ). This indicates that domains V C2591(N4) ¼ 2.9 A and II are in close contact and form an interaction chain through U2588, C765, G758, A764, A763 and G761 nucleotides. The one end of this chain (U2588) interacts with erythromycin while the other one (A764, A763 and G761) is in close contact with the L22 b-hairpin (Arg109-Gly112) (Figure 3(b)). As mentioned above, the other points of erythromycin interaction are G2484, A2045, A2042 and A2041 of domain V (Figure 4(a)). But A2041 inter˚ ). acts with U2000 (A2040(O1) – U2000(O2) ¼ 3.7 A The neighboring A2040 has contact with U1999 ˚ ) of IV– V inter(U1999(O3) –A2040(O3) ¼ 3.0 A domain region (Figure 4(a)), and this part of interdomain region, particularly A1997, A1996 and G1995, has strong interaction with less distorted part of the L22 b-hairpin (Figure 3(a)). Thus the L22 b-hairpin triplet deletion results in significant bending of the b-hairpin inside the tunnel that modifies the shape of its narrowest part formed by L4 and L22 (Figure 4(b)). The initial ˚ between a-chains of L4 and opening of about 12 A ˚ (side-chains are not L22 is reduced to 8.5– 9 A considered) due to bending of the b-hairpin tip. However, it would not inhibit the growth of the nascent polypeptide, because the polypeptide ˚ in diameter, and it seems also that it chain is , 6 A would not protect the ribosome from erythromycin binding either. It is known4 that erythromycin can bind to the resistant ribosomes without inhibiting the protein synthesis. Recent cryo-electron microscopy studies21 of the E. coli 70 S erythromycin-resistant ribosome with the L22 triplet deletion mutant help to explain that fact. It was demonstrated that the tunnel entry opening in the ribosome with the L22 mutant was twice that opening in the wt ribosome and was comparable with the opening in the initiator tRNA-complexed ribosome. It should be noted that the study21 also revealed the formation of an additional cavity between the beginning of the tunnel and the tip of protein L22, which would render the binding of erythromycin to the ribosome with the mutant L22 ineffective. It seems that this cavity is the space formed between the bent b-hairpin tip and the distorted 23 S rRNA domains II and V in Figure 4(a) and (b). This entirely agrees with the suggestion21 that the cavity in immediate vicinity to the L22 tip “might be formed as a result of mutation in the tip” and “it possibly involves reorganization in the central loop of domain V, induced by a transition in helix 35 of domain II”. It can be concluded now that the L22 triplet deletion mutation may lead to the following changes: it makes the side of the b-hairpin that is exposed to the tunnel surface highly hydrophobic
642
by removing polar or charged and strongly basic residues from the loop; it changes the overall orientation of the tip of the hairpin and disrupts its binding via Arg109-Gly112 to nucleotides G761, A763 and A764 of the 23 S rRNA domain II, which are in strong contact with U2588 of domain V—a binding point of erythromycin as reported;11 it bends the hairpin inwards the narrow part of the tunnel. A similar triplet deletion on the other side of the b-hairpin would be unfavorable. It may also put at risk the stability of whole assembly due to stronger interactions of that side with 23 S rRNA. The bending of b-hairpin weakens the contact between L22 and domain IV, however, residues 116– 119 in mutant L22 should still be very close to A1996-G1995 and the second interacting pocket of erythromycin via domain V (2041, 2042, 2045 and 2484), which is in contact with this part of domain IV, is less disturbed. Taking into account ˚ resolution) and conan approximate (at 3.5 A siderably different conformation of the 50 S bound erythromycin11 compare to its free structure26 and the dynamic behavior of the tunnel opening depending on the ribosome function21 we may speculate that there is also a possibility for the direct interaction between erythromycin and the A764 or C765 nucleotides exposed to the tunnel (Figure 4(a)). The whole undistorted erythromycin ˚ space molecule26 can be fitted well into , 15 A between the A764 and A2041 nucleotides. The nucleotides participating in erythromycin binding are located on opposite sides of the tunnel and are brought to those positions by interactions of domains II –V with L22. If there is mechanical blocking of polypeptide elongation by erythromycin as suggested by cryo-microscopy study,21 then it can be assumed that erythromycin, bound to the opposite walls of the tunnel, works as a gate interfering with further growth and progress of polypeptide after formation of a chain of 2 – 5 amino acid residues. Mutations in 23 S rRNA and/or in the L22 b-hairpin affect the orientation and distances between nucleotides, which participate in the binding of this antibiotic. Erythromycin, while still being able to bind to one of the sides of the tunnel, is unable to block the polypeptide growth in the drug-resistant ribosome.
Materials and Methods Protein expression, purification and crystallization The mutant protein was overexpressed, purified and crystallized as described earlier.12,28 Single plate-like 1.0 mm £ 0.5 mm £ 0.13 mm crystals have been obtained by the hanging drop vapor-diffusion technique. The drops were set up by mixing 2 ml of precipitant solution on siliconized cover slides and equilibrated against 1.0 ml of the same precipitant solution. Crystallization trays were incubated at 20 8C. The space group is P21, b ¼ 86:02 A; c¼ with cell dimensions a ¼ 31:79 A; b ¼ 104:38: 38:56 A;
Mutant L22 and Ribosome Resistance to Erythromycin
Data collection Selected crystals were mounted in thin-walled quartz capillaries, which were sealed with wax after filling both ends with reservoir solution. X-ray diffraction data were collected at room temperature using an MAR 345 Image Plate area detector (MarResearch, Hamburg, Germany) using X-rays from a Rigaku RU-200 rotating anode generator operating at 40 kV and 100 mA. X-rays were focused using nickel-coated mirrors. Due to crystal deterioration only 203 frames of 0.58 Dw oscillation were collected. However, it gave the completeness of 86%, which was enough to refine the structure to acceptable R-factors. The lack of actually observed data due to low intensity statistics is most noticeable in higher resolution shells (Table 1). Attempted low temperature data collection was less successful due to problems with even more rapid deterioration of protein crystals. Indexing, integration and scaling of data sets were carried out using DENZO and SCALEPACK.29 Structure determination The crystal structure was solved by the molecular replacement method using the CNS package.30 As a search model, a truncated version of water-free wt L22 (PDB accession number 1BXE13) structure was generated by removing the non-conserved b-hairpin region. The initial rotation search suggested that two molecules were in the asymmetric unit. The translation search was then used to determine which of the rotation solutions correspond to the two monomers. The final correlation coefficient of 0.577 and the packing value of 0.63 were significantly greater than those for the translation search with only one molecule. The positions of the two molecules A and B in the asymmetric unit were optimized to R ¼ 0.39 with the CNS30 rigid body leastsquares refinement using the whole available resolution range. Rfree cross-validation index based on randomly selected reflections (10% of the total set) was employed to monitor the subsequent refinement of the model. Following rigid body refinement, simulated annealing using torsion angle dynamics was used to improve the model. After that, b-hairpins for the two independent molecules were built into the generated density maps. Both sa-weighted 2mFo 2 DFc and mFo 2 DFc density maps30 were calculated and examined using the O program31 and XtalView.32 The electron density in the loop region of one molecule A was easily traced, while the electron density in the loop tip of the molecule B was highly diffused (residues 88 – 99), possibly as a result of molecule-packing difference. However, the latter was approximately traced and included in the model with ˚ 2). There was also a poor very high B-factors (70 – 116 A density in the C-terminal regions (residues 110 – 113) of both molecules. The side-chains were further adjusted stepwise during the course of refinement using restrained individual B-factor refinement followed by energy minimization of the coordinates. During the final stages of refinement, water molecules were inserted into the model only if there were peaks in the 2mFo 2 DFc maps higher than 2.0s with proper hydrogen-bonding distances. Water molecules with temperature factors ˚ 2 and real space correlation less than greater than 80 A 0.5 were excluded from subsequent steps of refinement. The final model containing 1738 protein non-H atoms, 37 solvent molecules gives an R factor of 20.9% (Rfree ¼ 25.5%). Assessment of the quality of the final structure showed that 91.4% of the residues falling in
643
Mutant L22 and Ribosome Resistance to Erythromycin
the most-favored region and 1% is in disallowed region of the PROCHECK Ramachandran plot.33 The latter residues are from the b-hairpin tip of the molecule B with poorly defined density. Further details of data collection, refinement statistics and final model quality are given in Table 1. All the Figures were generated using the program VMD.34 Accession number The coordinates for the mutant L22 have been deposited in the Brookhaven Protein Data Bank. The accession number is 1I4J.
Acknowledgments This work was supported by the Australian Research Council, the Russian Academy of Sciences and the Russian Foundation for Basic research. The research of M.B.G. was supported in part by the International Research Scholar’s award from the Howard Hughes Medical Institute.
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Edited by J. Doudna (Received 9 May 2002; received in revised form 15 July 2002; accepted 22 July 2002)