Báo cáo khoa học: Staphylococcus aureus elongation factor G – structure and analysis of a target for fusidic acid pdf

Staphylococcus aureus elongation factor G – structure and analysis of a target for fusidic acid Yang Chen, Ravi Kiran Koripella, Suparna Sanyal and Maria Selmer Department of Cell and Molecular Biology, Uppsala University, Sweden Introduction Protein synthesis, translation of mRNA into protein, is performed on the ribosome. To synthesize a protein, the ribosome goes through the phases of initiation, elongation, termination, and recycling, each phase being assisted by a number of protein translation fac- tors [1,2]. Some of these factors, in prokaryotes initia- tion factor 2, elongation factor Tu (EF-Tu), elongation factor G (EF-G), and release factor 3, are GTPases, which drive the process forwards using GTP as the energy source. Among these, only EF-G participates in two distinct steps of the translation cycle: elonga- tion and ribosome recycling. During elongation, after formation of each new peptide bond, EF-G binds to the ribosome and, under GTP hydrolysis, catalyses translocation, the concerted movement of mRNA, together with A-site and P-site tRNAs, to expose a new A-site codon [3,4]. Recycling takes place when the translating ribosome has reached a stop codon and released the nascent peptide. At this point, EF-G and ribosome recycling factor bind to the post-termination complex to catalyse the disassembly of the complex [5– 7]. EF-G has a low intrinsic activity in GTP hydrolysis that is stimulated by the interaction with the ribosome [8,9]. The currently prevalent model states that EF-G Keywords antibiotic resistance; crystallography; elongation factor G (EF-G); fusidic acid; switch region Correspondence M. Selmer, Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, 751 24 Uppsala, Sweden Fax: +46 18 536971 Tel: +46 18 4714177 E-mail: maria.selmer@icm.uu.se Database The atomic coordinates and observed structure factors are available in the Protein Data Bank database under the accession number 2XEX (Received 22 April 2010, revised 28 June 2010, accepted 14 July 2010) doi:10.1111/j.1742-4658.2010.07780.x Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) on the ribosome in a post-translocational state. It is used clinically against Gram-positive bacteria such as pathogenic strains of Staphylo- coccus aureus, but no structural information has been available for EF-G from these species. We have solved the apo crystal structure of EF-G from S. aureus to 1.9 A ˚ resolution. This structure shows a dramatically different overall conformation from previous structures of EF-G, although the indi- vidual domains are highly similar. Between the different structures of free or ribosome-bound EF-G, domains III–V move relative to domains I–II, resulting in a displacement of the tip of domain IV relative to domain G. In S. aureus EF-G, this displacement is about 25 A ˚ relative to structures of Thermus thermophilus EF-G in a direction perpendicular to that in previous observations. Part of the switch I region (residues 46–56) is ordered in a helix, and has a distinct conformation as compared with structures of EF-Tu in the GDP and GTP states. Also, the switch II region shows a new conformation, which, as in other structures of free EF-G, is incompatible with FA binding. We have analysed and discussed all known fusA-based fusidic acid resistance mutations in the light of the new structure of EF-G from S. aureus, and a recent structure of T. thermophilus EF-G in complex with the 70S ribosome with fusidic acid [Gao YG et al. (2009) Science 326, 694–699]. The mutations can be classified as affecting FA binding, EF-G– ribosome interactions, EF-G conformation, and EF-G stability. Abbreviations EF-G, elongation factor G; EF-Tu, elongation factor Tu; EM, electron microscopy; FA, fusidic acid; PDB, Protein Data Bank. FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS 3789 binds to the ribosome in GTP form, hydrolyses GTP, releases inorganic phosphate and, through a conforma- tional change, drives tRNA translocation [10] or ribo- some recycling [7]. However, there are recent indications that EF-G may act differently in transloca- tion and ribosome recycling [11]. The crystal structure of EF-G from Thermus thermo- philus was first solved in 1994 in complex with GDP [12] as well as in apo form [13]. Since then, structures of several mutants of EF-G from the same bacterium have been solved [14–16]. EF-G forms an extended structure consisting of five domains (Fig. 1A). The domain G (domain I) and domain II form a globular structure that is conserved in all other ribosomal GTPases. The exception is the additional subdomain G¢, which is inserted in domain G, and exists only in release factor 3 and EF-G. As in other GTPases, domain G contains a conserved P-loop, which coordinates the a-phosphate and b-phosphate, and two so-called switch regions, which coordinate the c-phosphate and change conformation between a tense GTP state and a relaxed GDP state [17]. Ribosomal complexes that have been stalled by the locking of EF-G to the ribosome with either a nonhy- drolysable GTP analogue [18] or the antibiotic fusidic acid (FA) and GDP [19–21] have been visualized with cryo-electron microscopy (EM) and single-particle reconstructions. Recently, a 3.6 A ˚ crystal structure of EF-G bound to the T. thermophilus ribosome showed the FA-binding site for the first time, and revealed the detailed interactions of EF-G with the ribosome [22] (summarized in Fig. 1B). As compared with this FA- stabilized, ribosome-bound conformation, crystal struc- tures of T. thermophilus EF-G display different confor- mations, where domains III–V have rotated relative to domains I–II, resulting in the position of the tip of domain IV differing by 20 A ˚ (Fig. 2; discussed further below). It appears that ribosome binding is the main trigger of the conformational change in EF-G, as in solution it can accommodate GDP or GTP without forcing any major changes in its global conformation [15,23,24]. FA is a clinically used steroid antibiotic that locks EF-G on the ribosome after GTP hydrolysis and trans- location [25]. FA binds to a pocket between domains I, II and III of EF-G, and seems to lock EF-G in a conformation intermediate between the GTP-bound and GDP-bound forms [22]. Staphylococcus aureus is one of the major clinical targets for FA treatment. However, very few studies have been performed using EF-G from this species. In this study, we have solved the apo crystal structure of S. aureus EF-G to 1.9 A ˚ resolution, allowing us to examine the generality of conclusions drawn from the T. thermophilus EF-G structures and to pinpoint the role of amino acids that are mutated in isolated FA-resistant strains of S. aureus [26,27]. Results and Discussion Structure solution S. aureus EF-G was crystallized in a mixture of poly- ethylene glycol 3350 and NaCl in Tris ⁄ HCl buffer at pH 8.7. The crystals grew in space group P2 1 and diffracted to 1.9 A ˚ resolution (Table 1). There are two molecules in the asymmetric unit, forming a noncrys- tallographic two-fold symmetry. b-Sheets from domain V of molecules A and B form an extended b-sheet, and helix A V packs in an antiparallel fashion to the equivalent helix in molecule B. Residues 2–38, 64–441 and 445–692 in molecule A and residues 2–41, 46–56, 65–441 and 447–692 in molecule B were ordered and could be built into the electron density maps. The ordered part includes domain III, which is disordered in structures of wild-type T. thermophilus EF-G [12,13]. In molecule B, part of the switch I region could be built. The entire switch I region is disordered in all previous EF-G structures from T. thermophilus [12–16], including the EF-G–70S complex structure with GDP and FA [22], and has been suggested to be ordered only in the ribosome-bound GTP state, as Table 1. Summary of crystallographic data and refinement. Data collection statistics Resolution (A ˚ ) a 46.2–1.9 (2.0–1.9) R meas [48] (%) 6.1 (55.5) I ⁄ r I 16.5 (3.1) Completeness (%) 92.7 (78.5) Redundancy 3.71 (3.56) Refinement statistics Resolution (A ˚ ) 46.2–1.9 Number of unique reflections ⁄ test set 110 987 ⁄ 4777 R work ⁄ R free (%) 18.7 ⁄ 22.4 Molecules per asymmetrical unit 2 Number of atoms Protein 10 352 Water 509 Ions 4 Average B-factor (A ˚ 2 ) 22.4 Rmsd from ideality Bond lengths (A ˚ ) 0.020 Bond angles (°) 1.53 Ramachandran statistics Residues in most favoured regions (%) 96.75 Residues in additional allowed regions (%) 2.87 Residues in disallowed regions (%) 0.38 a Values in parentheses represent the highest-resolution bin. Crystal structure of Staphylococcus aureus EF-G Y. Chen et al. 3790 FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS indicated by recent proteolytic cleavage experiments [28]. It is also disordered in structures of the eukary- otic equivalent, eEF2 [29]. In the present structure, res- idues 46–56 form a helix that was only clearly visible in difference Fourier maps after refinement of the rest of the structure. The density for residues 42–45 was too weak to allow interpretation, but this region would need to be in an extended conformation to bridge 16.5 A ˚ between the a carbons of residues 41 and 46. In molecule A, the density for the entire switch I region is too weak for interpretation, indicating higher flexibility. Attempts to soak S. aureus EF-G crystals with GDP as well as nonhydrolysable GTP analogues resulted in partial occupancy of GDP in the nucleotide-binding site. Therefore, we present here the apo structure of S. aureus EF-G. Overall structure and comparison with previous EF-G structures All five domains of S. aureus EF-G are ordered in our structure. The overall conformation of the two EF-G molecules in the asymmetric unit is very similar (rmsd of 0.58 A ˚ for 660 C a atoms), with only a slight differ- ence in the orientation of domains III and IV. Thus, when the two molecules are superimposed on the basis of domains I and II, the maximum difference at the edge of domain III is 2.3 A ˚ . Comparison of S. aureus EF-G with the previously solved T. thermophilus EF-G structures shows that the individual domains are highly similar. However, domains III, IV and V are in a different orienta- tion relative to domains I and II in comparison to previous EF-G structures (Fig. 1C). Between all the A B C Fig. 1. EF-G structure. (A) Overall structure and structural domains of S. aureus EF-G (PDB 2xex). The switch regions are shown in black, with switch II facing domains II and III, and switch I behind the G-domain. (B). Crystal structure of EF-G bound to the T. thermophilus 70S ribosome with GDP and FA (PDB 2wri [22]). FA (left) and GDP (right) are shown in black. Numbers indicate ribosomal contact areas: 1, decoding centre; 2, 23S RNA 2475 loop; 3, 23S RNA 1067 ⁄ 1095 loops; 4, ribosomal protein L6; 5, C-terminal domain of ribosomal protein L12; 6, 23S RNA 2660 loop (from the back); 7, ribosomal protein S12. Thickness of lines indicates closeness to the viewer. (C) Comparison of apo-EF-G from S. aureus (PDB 2xex, magenta) and T. thermophilus (PDB 1elo [13], grey). Superposition is based on domains I and II. Y. Chen et al. Crystal structure of Staphylococcus aureus EF-G FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS 3791 T. thermophilus EF-G structures, wild type and various mutants, domains III, IV and V display a movement relative to domains I and II, resulting in a shift of the tip of domain IV of up to 8 A ˚ [14,16]. The ribosome- bound structure of EF-G as visualized by crystallogra- phy [22] shows a larger movement in the same direc- tion, measuring about 27 A ˚ . The equivalent comparison with S. aureus EF-G shows a shift of domains III, IV and V by approximately 25 A ˚ in the perpendicular direction (Fig. 2). The hinge region for this conformational change consists of residues 400– 405, as previously observed in T. thermophilus EF-G [16]. Conformational changes of EF-G in more than one direction were suggested in early cryo-EM studies of ribosome–EF-G complexes [20], but because of the low resolution (17–20 A ˚ ), the structural interpretation is not very reliable. The P-loop (residues 12–27) has the same conforma- tion as in the apo structure of T. thermophilus EF-G [13], and upon crystal soaking with GDP, partial occu- pancy of the peptide-flipped structure is observed, in agreement with structures of T. thermophilus EF-G [30] (data not shown). The switch I region consists of residues 39–63. The ordered part is a helix from residues 46 to 56 that packs against helix A G so that Trp52 makes hydropho- bic interactions with Leu31, Tyr32 and Ile37, and Met53 interacts with Glu28 (Fig. 3A). With the exception of Ile37, all of these residues are conserved in EF-G from different species. In contrast to the situation in EF-G, the switch I region is fully ordered in structures of EF-Tu with GDP [31] and a GTP analogue [32], as well as in the structure of the EF-G homologue EF-G-2 with GTP [18]. In EF-Tu, the switch I region forms a short helix followed by a b-hair- pin reaching away from the nucleotide-binding site in the GDP state, whereas in the GTP state, it forms two short helices just before the conserved Thr that coordi- nates a magnesium ion and the c phosphate. The helix that we observe is longer than in any of these structures, and has a different orientation (Fig. 3B). It is too far away from the nucleotide-binding site to allow the inter- action of the conserved Thr62 with magnesium and c phosphate that should occur in the GTP state. Superpo- sition of the current structure with the ribosome-bound EF-G [22] shows that the observed switch I conforma- tion would be compatible with ribosome binding and located in the intersubunit space at a distance of 10 A ˚ from residue 2655 of 23S RNA, 15 A ˚ from ribosomal protein L14, and 10 A ˚ from residue 342 of 16S RNA. The switch II region of S. aureus EF-G has electron density for all residues, including side chains, except for Gly84 (Fig. 3C). It has a different conformation compared to the T. thermophilus EF-G structures (Fig. 3D). The hydrogen bond Asp87–Arg659 stabi- lizes the switch II region and the current domain 38 Å ABC Fig. 2. Conformational space of EF-G on and off the ribosome. (A) Superposition of S. aureus EF-G with the ribosome-bound T. thermophilus EF-G (PDB 2wri [22], dark blue) and the T. thermophilus apo-EF-G structure (PDB 1elo [13], yellow), based on domains I and II. The arrow indicates the direction of projection to the circle in (C). (B) As (A), view 90° away. (C) Comparison of the positions of the tip of domain IV in all available EF-G structures in the PDB. The structures were superimposed on the basis of domains I and II. Looking from the direction of the arrow in (A) and (B), the coordinates of His572 at the tip of domain IV are roughly in one plane, and were manually covered with a col- oured dot. 1, E. coli EF-G + GMPPNP + 70S (PDB 2om7 [18], cryo-EM); 2, T. thermophilus EF-G + 70S + FA + GDP (PDB 2wri [22]); 3, E. coli EF-G + GDP + 70S + FA (PDB 1jqm [21], cryo-EM); 4, T. thermophilus EF-G T84A + GMPPNP (PDB 2bv3 [15]); 5, T. thermophilus EF-G + GDP (dimer, PDB 1ktv); 6, T. thermophilus EF-G apo (PDb 1elo [13]); 7, T. thermophilus EF-G T84A + GDP, FA-resistant (PDB 2bm0 [14]); 8, T. thermophilus EF-G G16V + GDP, FA-hypersensitive (PDB 2bm1 [14]); 9, T. thermophilus EF-G H573A + GDP (PDB 1fnm [16]); 10, S. aureus EF-G (PDB 2xex). Crystal structure of Staphylococcus aureus EF-G Y. Chen et al. 3792 FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS A C B D E Fig. 3. The switch regions of EF-G. (A) Switch I region of S. aureus EF-G. The ordered switch I helix packs against helix A G . The 2F o )F c map is contoured at 1r. (B) Comparison of switch I in EF-G and EF-Tu. The structures were superimposed on the basis of the equivalent parts of domains G and II. The switch I region of S. aureus apo-EF-G (PDB 2xex, magenta), EF-Tu in complex with GDP (PDB 1tui, yellow [31]) and EF-Tu in complex with GDPNP (PDB 1eft, green, Mg 2+ , green sphere and GDPNP, in cpk [32]) are shown together with the EF-G structure (grey). The first, shorter helix shown in green is identical in the GDP and GTP forms of EF-Tu. (C) F o )F c omit map of the switch II region of S. aureus EF-G contoured at 3r. Omitted residues are shown in yellow stick representation. (D) Comparison of the switch II region from different EF-G crystal structures. Superposition based on helix B G (Val90-Asp100) of S. aureus EF-G (PDB 2xex, magenta); T. thermo- philus EF-G wild type (PDB 1elo, yellow [13]); T. thermophilus EF-G H573A (PDB 1fnm, orange [16]); T. thermophilus EF-G T84A (PDB 2bm0, light blue [14]); T. thermophilus EF-G G16V (PDB 2bm1, red [14]); T. thermophilus EF-G T84A with GDPNP (PDB 2bv3, green [15]); T. thermophilus EF-G–GDP–FA complex with the ribosome (PDB 2wri, dark blue [22]). Residues 20–200 are shown, but only the switch II region and the side chain of Phe88 are coloured. (E) Domain III and the FA-binding site. Switch II regions of S. aureus EF-G (magenta) and FA-bound EF-G at the ribosome (PDB 2wri [22], switch II in dark blue, FA in yellow ⁄ red) superposed on the basis of domain III (grey, residues 407–474), showing how the FA-binding site in the S. aureus EF-G structure is blocked by the switch II region. Y. Chen et al. Crystal structure of Staphylococcus aureus EF-G FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS 3793 arrangement, with a larger contact area between domains II and V than that in any other structure of EF-G. The switch II region and the conserved Phe88 (Phe90 in T. thermophilus) displays many different ori- entations in the available crystal structures, and none of the isolated EF-G structures displays a switch II conformation identical to that of EF-G in complex with the 70S ribosome and FA [22] (Fig. 3D). Whereas Phe88 in the ribosome-bound structure is exposed at the surface of EF-G and forms part of the FA-binding site, it points to the opposite direction in our structure, interacting with Glu93 in helix B G and Tyr126 in helix C G in the core of the domain, and blocking the FA- binding site (Fig. 3E). Despite its many different con- formations, the switch II region also blocks the FA- binding site in all structures of free T. thermophilus EF-G (not shown). However, we do not believe that the alternative switch II conformations observed in structures of wild-type and mutant T. thermophilus EF-G are responsible for FA sensitivity and resistance, respectively [14]. Rather, the switch II region only adopts its FA-stabilized conformation when bound to the ribosome in the presence of the drug, and several FA resistance mutations in the switch II region influ- ence direct contacts with FA (discussed further below). Conformational space of EF-G The present structure of S. aureus EF-G shows that EF-G, when not bound to the ribosome, can acquire AB CD Fig. 4. FA resistance mutations. (A) All known FA resistance mutation sites (Table 2) mapped on the S. aureus EF-G structure. The mutation sites are displayed as side chains and located in domain III, domain V and the interface of domains G, III and V. (B) Mutation sites in domain III that may affect the FA-binding pocket. Mutation sites are shown with yellow carbons, and influence the packing between helix A III and helix B III . To the left is the interface with domains I, II and V; to the right is the connection to domain IV. (C) Mutation sites in helices A V and B V at the surface of domain V. Gly621 and Gly617 are in the area of contact with the 1095 and 2473 regions of 23S RNA. The two helices are facing the ribosome, and the four-stranded b-sheet is facing the other domains of EF-G. (D) Linker region between domains I–II and domains III–V. The four sites of FA resistance mutations in this region are shown with side chains. Crystal structure of Staphylococcus aureus EF-G Y. Chen et al. 3794 FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS a conformation that is distinctly different from what has previously been observed for T. thermophilus EF-G. The fact that the two molecules in the asym- metric unit show identical conformations, despite being involved in different crystal contacts, suggests that this is not a crystallographic artefact. In the many different crystal structures of T. thermo- philus EF-G, only smaller conformational differences have been observed [12–16]. An explanation for this could be that the interdomain movements of T. ther- mophilus EF-G are limited by crystal contacts between domains G¢ and IV, which form an extended b-sheet between the two domains [15,33]. By crystallizing EF-G from another species, S. aureus, we have obtained a new crystal packing arrangement that avoids this problem. Furthermore, the only existing data on the confor- mation of EF-G in solution come from small-angle scattering measurements on T. thermophilus EF-G [23]. These measurements resulted in radii of gyra- tion in the range 30.2–32.9 A ˚ for EF-G bound to different nucleotides, and the difference between the different states was judged to be nonsignificant as compared with the experimental errors. The calcu- lated radius of gyration from the S. aureus EF-G crystal structure is 30.6 A ˚ , whereas the corresponding value for T. thermophilus EF-G [Protein Data Bank (PDB) 1fnm] [16] is 30.5 A ˚ , agreeing equally well with those measurements. In conclusion, EF-G may display larger interdomain flexibility in solution than previously thought. Our new conformation is signifi- cant, as it demonstrates the size of the conforma- tional space of EF-G when not bound to the ribosome. The active conformation of EF-G is the one that occurs on the ribosome. So far, only post-transloca- tional states, where domain IV of EF-G has entered the A-site, have been visualized on the ribosome [18–22]. The ribosome-bound EF-G conformations in the presence of GMPPNP or GDP and FA differ by approximately 6 A ˚ in position of the tip of domain IV when the G-domains are superimposed (Fig. 2C, points 1 and 2). However, there is, at present, no structural information regarding the initial binding of EF-G to a presumably ratcheted ribosome where the 30S A-site is still occupied by the peptidyl tRNA. Most likely, ribosome binding induces a somewhat stable but transient conformation of EF-G that is compatible with a tRNA in the 30S A-site, and we can only speculate that this conformation of EF-G may be more similar to either of the confor- mations observed in the crystal structures of free EF-G. FA resistance mutations FA binds to EF-G on the ribosome and prevents its dissociation after GTP hydrolysis and translocation. In the recent crystal structure of a 70S–EF-G complex [22] (Fig. 1B), it is shown that FA allows the switch I region to change from a GTP to a GDP conformation, whereas the switch II region is prevented from adopt- ing its GDP conformation. This, in turn, stops the glo- bal conformational change of EF-G to the GDP state that would leave the ribosome. In other words, FA locks EF-G in a conformation between its GTP and GDP forms that cannot dissociate from the ribosome. FA resistance mutations belong to three classes: fusA mutants, with mutations in the EF-G gene; fusB, fusC and fusD mutants, which express a resistance pro- tein that somehow protects the cell from FA inhibi- tion; and fusE mutants, with mutations in ribosomal protein L6 [27]. There are, in total, 42 positions in EF-G where point mutations of the fusA class have been reported to cause FA resistance [27,34,35] (Fig. 4A). The previ- ous analysis of these [16] was performed without accu- rate knowledge of the FA-binding site [22], and, in addition, new mutations have recently been identified [27]. On the basis of analysis of the S. aureus EF-G structure together with the recent T. thermophilus EF- G–70S complex structure with FA [22], we can now classify the mutations into four groups, A–D (Table 2). These perturb four critical parameters for locking EF- G to the ribosome: drug binding (A), ribosome–EF-G interactions (B), EF-G conformation (C), and EF-G stability (D). Several mutations seem to affect more than one of these parameters; for example, EF-G con- formation and stability are intimately linked to FA binding as well as ribosome binding. Group A mutations involve residues in direct con- tact with FA as well as residues that shape the drug- binding pocket. These resistance mutations will directly alter drug–EF-G interactions, probably lowering the affinity of FA for the ribosome-bound EF-G. The switch II loop directly contributes to the FA-binding site, where both Thr82 and Phe88 are in direct contact with FA in the ribosome complex structure [22]. Muta- tion of the corresponding residues in T. thermophilus (Thr84 and Phe90) also leads to resistance [36]. One edge of the FA-binding pocket is formed by domain III [22]. A cluster of mutation sites is located in this area, where the C-terminal end of helix A III packs against the central part of helix B III (Fig. 4B). Asp434 and Thr436 in helix A III both form hydrogen bonds with His457 in helix B III . Thus, the mutations P435Q, T436I, H457Y and P435Q will change this Y. Chen et al. Crystal structure of Staphylococcus aureus EF-G FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS 3795 Table 2. Structural interpretations of all known FA resistance mutations. Residues are numbered according to the S. aureus EF-G sequence. Helices are alphabetically ordered within each domain, and labeled with the domain subscript. b-Strands are numbered within each domain, and labeled with the domain subscript. A, mutations influence the FA-binding site; B, muta- tions influence EF-G–ribosome interactions; C, mutations influence interdomain interactions in EF-G; D, mutations influence the stability of EF-G. Residue Species Mutation Group Location Interpretation of mutation Ala66 E. coli [34,49] Val C Domain G, at the C-terminal end of the switch I region, at the interface to domain II Mutation can push apart domains I and II or possibly affect the switch I conformation Thr82 Salmonella typhimurium [34] T. thermophilus [36] Ala A Domain G, in the switch II region. In the ribosome complex structure, at the interface of domain G and domain II, and contacts FA [22] Mutation affects the FA-binding site [22] Phe88 S. aureus [35] T. thermophilus [36] Leu A Domain I, in the switch II region. In the ribosome complex structure, in contact with FA [22] Mutation affects the FA-binding site [22] Val90 a S. aureus [35] Ile C Domain G, in the switch II region. The equivalent position in T. thermophilus, S. typhimurium and E. coli is Ile. In T. thermophilus complex [22], packing against domain III Mutation may affect domain I–III interactions Ala102 S. typhimurium [34] Glu D Domain G, in b-strand 5 G . Packing against Met16 Mutation will induce steric hindrance, and may disturb the structure of domain G Leu106 a S. typhimurium [34] Ser D Domain G, in b-strand 5 G . Involved in hydrophobic interactions with Val112, Val132 and Leu151. Phe in T. thermophilus, Tyr in Mutation can disturb the hydrophobic core of domain G Pro114 S. aureus [27] His B, C Domain G, in the turn before helix C G , at the interface with domain V. Packing against Gly664. In the ribosome complex [22], close to 2660 of 23S RNA Mutation would change conformational properties, and may influence the domain G–V interaction as well as ribosome interactions Gln115 S. aureus [27,35] E. coli [49] Leu B Domain G, in helix C G , hydrogen bonding to P-loop His18. In the ribosome complex [22], hydrogen bonds to His85 and Thr118, close to 2660 of 23S RNA Mutation will probably affect the ribosome-binding surface Thr118 S. typhimurium [34] Ile B, C Domain G, in helix C G , at the interface with domain V. In the ribosome complex [22], hydrogen bonds to His85 in switch II, close to 2660 of 23S RNA Mutation may influence the domain G–V interaction as well as ribosome interactions Val119 S. typhimurium [34] Leu D Domain G, in helix C G , packing against Met16 in the P-loop. In the ribosome complex [22], contacting Val104 Mutation will make a slight change to the core of domain G Gln122 S. typhimurium [34] His C Domain G, hydrogen bonding to Thr667 in domain V; close to Phe88. This interaction is not present in the ribosome complex [22] Mutation may affect interactions with domain V Val132 a S. typhimurium [34] Thr D In the hydrophobic core of domain G, close to Leu106, Leu151 and Val256. Ala in T. thermophilus and S. typhimurium Mutation may disturb the hydrophobic core of domain G Leu155 S. typhimurium [34] Pro C, D At the C-terminal end of helix D G , hydrophobic interaction swith Trp120 in helix C G [16]. Close to the interface with domain V. This interaction is not present in the ribosome complex [22] Mutation will break the helix and may affect interactions with domain V Crystal structure of Staphylococcus aureus EF-G Y. Chen et al. 3796 FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS Table 2. (Continued). Residue Species Mutation Group Location Interpretation of mutation Thr385 S. aureus [27] Asn C Domain II, packing against the C-terminal end of helix A III in domain III. This interaction is not present in the ribosome complex [22] Mutation could influence the domain II–III interaction Pro404 S. aureus [27,35] Leu Arg Gln C In the linker region between domains II and III, the main hinge region for conformational change of EF-G Mutation will influence the linker conformation, which could affect the relative orientation between domains I, II and III and thereby the FA-binding site Pro406 S. aureus [27,35] S. typhimurium [34] Leu C In the linker region between domains II and III, the main hinge region for conformational change of EF-G Mutation will influence the linker conformation, which could affect the relative orientation between domains I, II and III and thereby the FA-binding site Val407 S. aureus [35] Phe C Domain III, at the interface with domain V. Packing against the linker to domain IV. In the ribosome- bound state [22], this linker has flipped away to create the 40 A ˚ shift of domain IV relative to domain III Mutation to a larger side chain may change the relative positions of domain III and V [16] as well as the position of domain IV in the free state Ala426 S. typhimurium [34] Asp B, D Domain III, in the middle of helix A III in the hydrophobic core. In the ribosome complex [22], this helix binds to 16S RNA and S12 Mutation to a large, nonhydrophobic residue leads to a steric clash, lowers the stability of domain III, and may affect nearby ribosome interactions Leu430 S. typhimurium [34] Gln B, D Domain III, in the middle of helix A III in the hydrophobic core. In the ribosome complex [22], this helix binds to 16S RNA and S12 Mutation to a nonhydrophobic residue lowers the stability of domain III and may affect nearby ribosome interactions Asp434 S. aureus [27,35] Asn A Domain III, at the C-terminal end of helix A III . Hydrogen bonding to His457 in helix B III . In the ribosome complex, in contact with FA [22] Mutation would affect the surface in the FA-binding pocket Pro435 S. typhimurium [34] Gln A Domain III, in a turn after helix A III . In the ribosome complex [22], the previous residue is in contact with FA Mutation will change the turn conformation, affecting the interactions of Asp434 and Thr436 and the FA- binding site Thr436 S. aureus [27,35] Ile A Domain III, in the turn after helix A III . Hydrogen bonding to His457 in helix B III . In the ribosome complex, lining the FA-binding site [22] Mutation would affect the surface in the FA-binding pocket His438 a S. aureus [27] Asn C Domain III, in the turn after helix A III . Packing against Pro406 in the linker between domains II and III. Arg in T. thermophilus Mutation may influence the linker conformation and relative orientation between domains I, II and III Gln447 S. typhimurium [34] His C, D Domain III, at the C-terminus of the loop in the b- sheet; hydrogen bonding with Ser411 in next strand. In the ribosome complex structure [22], in the area of contact with the linker to domain IV Mutation may affect interdomain interactions Gly451 a S. aureus [35] Val C, D Domain III, packing against Pro406 in the linker to domain IV. Ser in T. thermophilus Side chain may influence domain III stability and ⁄ or interdomain interactions Gly452 S. aureus [27,35] Ser Cys Val A, C, D Domain III, close to helix B III and to the linker to domain IV Side chain would clash with His457 in helix B III , and may affect the FA-binding pocket. May push His457 to the interface of domain G and III [16] Y. Chen et al. Crystal structure of Staphylococcus aureus EF-G FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS 3797 Table 2. (Continued). Residue Species Mutation Group Location Interpretation of mutation Met453 S. typhimurium [34] Ile A Domain III, in the turn before helix B III at the interface with domains G and V. In the ribosome complex structure, in hydrophobic interactions with Phe88 [22] Mutation probably affects the FA-binding pocket Leu456 S. aureus [27,35] Phe A, B Domain III, at the interface with domains G and V. In the ribosome complex [22], in contact with A2662 of 23S RNA and lining the FA-binding pocket Mutation may affect FA binding and ⁄ or ribosome interactions His457 S. aureus [27,35] Tyr A Domain III, in helix B III ; forming hydrogen bonds to Thr436 and Asp434; stabilizing domain III. In the ribosome complex [22], lining the FA-binding site Mutation would affect the surface in the FA-binding pocket Leu461 a S. aureus [35] Ser A, D Domain III, in helix B III ; involved in hydrophobic interactions with Leu430, Phe437 and Ile450. Ile in T. thermophilus. In the ribosome complex [22], the following residue packs against FA Mutation may change the position of helix B III and affect FA binding Arg464 S. aureus [27,35] S. typhimurium [34] Ser His Leu Cys A Domain III, in helix B III ; making hydrogen bonds with Glu433 in helix A III . In the ribosome complex [22], contributing to the FA-binding pocket Mutation probably affects the FA-binding pocket Gly617 S. aureus [27] Asp B Domain V, helix A V , contacts second molecule. In the ribosome complex [22], packing against A1095 of 23S RNA. The next residue interacts with L6 Mutation may disturb ribosome interactions Gly621 S. typhimurium [34] Cys B Domain V, helix A V , contact with second molecule. In the ribosome complex [22], packing against U2473 of 23S RNA Mutation may disturb ribosome interactions Gly628 S. aureus [27] Val B, D Domain V, at the N-terminus of strand 2 V , pointing towards the neighbouring b-strand 3 V . In the ribosome complex, close to interaction with U2473 of 23S RNA [22] Mutation to introduce a side chain would induce a steric clash and conformational change, and affect the nearby ribosome contact Gly632 S. typhimurium [34] Asp B Domain V, in the b-strand that packs with a second molecule. In the ribosome-bound structure, the previous residue interacts with 1067 of 23S RNA [22] Mutation will change the domain V surface, probably affecting ribosome interactions Pro647 S. typhimurium [34] Gln C Domain V, at the interface with domain IV. The neighbouring residue 648 packs against domain III. This interaction between domains III, IV and V is conserved in the ribosome-bound structure [22] Mutation will change the backbone conformation and create a steric clash, affecting the interdomain interactions Ala655 S. aureus [27] Glu C Domain V, in helix B V at the interface with domain G. In the ribosome complex, packing against domain III [22] Mutation to a larger side chain would disrupt the interaction and may influence the domain arrangement or shift the position of helix B V , thereby influencing ribosome interactions Crystal structure of Staphylococcus aureus EF-G Y. Chen et al. 3798 FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... elongation factor EF-Tu-GDP Structure 4, 114 1–1 151 Kjeldgaard M, Nissen P, Thirup S & Nyborg J (1993) The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation Structure 1, 3 5–5 0 Liljas A, Kristensen O, Laurberg M, Al-Karadaghi S, Gudkov A, Martemyanov K, Hughes D & Nagaev I (2000) The states, conformational dynamics, and fusidic acid- resistant mutants of elongation. .. Biological cost and compensatory evolution in fusidic acid- resistant Staphylococcus aureus Mol Microbiol 40, 43 3–4 39 Martemyanov KA, Liljas A, Yarunin AS & Gudkov AT (2001) Mutations in the G- domain of elongation factor G from Thermus thermophilus affect both its interaction with GTP and fusidic acid J Biol Chem 276, 2877 4–2 8778 Li JJ, Venkataramana M, Wang AQ, Sanyal S, Janson JC & Su ZG (2005) A mild... sensitivity in EF -G J Mol Biol 348, 93 9–9 49 Hansson S, Singh R, Gudkov AT, Liljas A & Logan DT (2005) Crystal structure of a mutant elongation factor G trapped with a GTP analogue FEBS Lett 579, 449 2–4 497 Laurberg M, Kristensen O, Martemyanov K, Gudkov AT, Nagaev I, Hughes D & Liljas A (2000) Structure of a mutant EF -G reveals domain III and possibly the fusidic acid binding site J Mol Biol 303, 59 3–6 03 Vetter... during data collection, and K Backbro and C S ¨ Koh for comments on the manuscript This work was supported by individual grants from the Swedish Research Council, the Wenner Gren foundation and Crystal structure of Staphylococcus aureus EF -G Carl Trygger’s Foundation to M Selmer and S Sanyal, the Goran Gustafsson Foundation to S Sanyal, ¨ and Magnus Bergvall’s foundation and the Swedish Foundation for. .. physical connection between domains G and II and domains III, IV and V In the S aureus EF -G structure, this loop has a bent conformation, and packs against domain III and interacts with the linker between domains III and IV (Fig 4D) Pro404 and Pro406 are critical for this conformation His438, within hydrogen-bonding distance of Glu405 and the carbonyl group of Pro404, also contributes to this particular... 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Staphylococcus aureus elongation factor G – structure and analysis of a target for fusidic acid Yang Chen, Ravi Kiran Koripella, Suparna Sanyal and Maria. 93 9–9 49. 15 Hansson S, Singh R, Gudkov AT, Liljas A & Logan DT (2005) Crystal structure of a mutant elongation fac- tor G trapped with a GTP analogue.