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;

˚
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

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

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

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

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

[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

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

˚
, 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

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

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

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-

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-

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

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

ated at the N-terminal end of helix C
G
. The mutations
P114H, Q115L and T118I will modify the surface of
EF-G exposed for interaction with the 2660 region of
23S RNA.
Several group B mutation sites occur in helices A
V
and B
V
at the surface of domain V (Fig. 4C). Helix A
V
is packed against the second molecule in our structure,
whereas in the structure of EF-G in complex with the
ribosome [22], this helix interacts with the 1095 and
2473 regions of 23S RNA. Helix B
V
packs against
domains G and III, and in complex with the ribosome
the C-terminal turn of the helix (659–663) interacts
with the 2660 region of 23S RNA as well as with
the C-terminus of ribosomal protein L6 [22]. Resis-
tance mutations in this region presumably either
disturb helix B
V
(S660P) or the following turn
(G664A ⁄ G664S), or make changes to the outer surface
of the helix (A655E, R659C ⁄ R659H ⁄ R659S), affecting
the surface in contact with the ribosome. The contacts
of domain V with domains G and III undergo large

Ala
B, C Domain V, at the interface with domain G, in contact
with Pro114. In the ribosome complex [22], this
interface changes; now close to the interaction
with 2660 of 23S RNA. Backbone conformation not
accessible for other residues
Mutation will change the backbone conformation,
may influence the domain G–V interaction as well
as ribosome interactions
Gly666 S. aureus [27] Val C Domain V, in strand 4
V
, at the interface with
domain G
Mutation may influence the domain G–V interaction
Met670 S. typhimurium [34] Ile C Domain V, in strand 4
V
, at the interface with
domain III
Mutation may influence the domain III–V interaction
a
Residue is not conserved between S. aureus and T. thermophilus.
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 3799
The only ribosomal mutations conferring resistance
against FA occur as truncations or frameshift muta-
tions in ribosomal protein L6 [27]. Probably, they have
an effect similar to the group B mutations of EF-G,
because, in the FA complex of EF-G with the ribo-
some, the C-terminus of ribosomal protein L6 is in
direct contact with domain V of EF-G [22].

G
in domain G, and the
C-terminal part interacts with the turn before helix B
III
in domain III. The G664A, G666V and M670I muta-
tions could possibly affect these interactions. In the
S. aureus structure, the T118I mutation in domain G
would affect the same interaction, and in domain III,
the 438 region, containing several mutation sites, is
close in space. Both interactions of this b-strand
change upon ribosome binding (compare PDB 2wri
[22]).
The switch II mutation V90I is puzzling, as the addi-
tion of a single methyl group in S. aureus provides
32-fold FA resistance [27], whereas several other
species, such as T. thermophilus, have Ile in the
corresponding position without being resistant. In the
FA-locked conformation of T. thermophilus EF-G [22],
Ile92 is packed against domain III. As the directly
interacting residues in domain III are conserved in
S. aureus, the resistance is probably caused by the resi-
due at position 92 in combination with the sequence
variation at the surface of domain III that interacts
with domain I, motivating the classification of this
mutation as group C.
Finally, group D mutations seem to decrease the
structural stability of domains I and III of EF-G. This
is probably another way of preventing the locking of
EF-G, and these mutants with lower stability may dis-
sociate faster than the wild type from the ribosome,

–EF-G was
immediately dialysed against buffer A. Further purification
was performed on a Hiload 16 ⁄ 60 Superdex-75 (GE Health-
care) gel filtration column equilibrated with buffer A. The
purified protein was concentrated to 4 mgÆmL
)1
with an Am-
icon Ultra 10 kDa cut-off centrifugal filter unit (Millipore,
Billerica, MA, USA) and stored at )80 °C after shock freez-
ing in liquid nitrogen. Before crystallization, the buffer was
exchanged for 20 mm Tris ⁄ HCl (pH 7.5) and 200 mm NaCl
in a 10 kDa cut-off Vivaspin concentrator.
Crystallization
Initial microcrystals of S. aureus EF-G grew in the Index
screen (Hampton Research, Aliso Viejo, CA, USA) with a
reservoir solution containing 100 mm Tris ⁄ HCl (pH 8.5),
200 mm NaCl and 25% (w ⁄ v) poly(ethylene glycol) 3350.
In attempts to reproduce these crystals, they appeared only
Crystal structure of Staphylococcus aureus EF-G Y. Chen et al.
3800 FEBS Journal 277 (2010) 3789–3803 ª 2010 The Authors Journal compilation ª 2010 FEBS
after 1 month and in few of the drops. Therefore, S. aureus
EF-G crystals were grown in streak-seeded sitting drop
vapour diffusion experiments. Two microlitres of a
4mgÆmL
)1
protein solution was mixed with 2 lL of reser-
voir solution [20–25% (w ⁄ v) poly(ethylene glycol) 3350,
200 mm Tris ⁄ HCl, pH 8.7, 200 mm NaCl] and equilibrated
against 400 lL of reservoir solution at 295 K. Crystals
appeared after 1–2 weeks, and grew to 100–150 l m in size

phaser [39], using domains I and II from T. thermophilus
EF-G as a search model (PDB 1fnm [16]). Domains V, III
and IV were sequentially placed in the difference maps with
coot [40]. The structure was improved by the use of auto-
matic model building in arp ⁄ warp [41]. Further structure
rebuilding was performed in coot [40], and the structure
was refined, with cns [42,43] and refmac [44,45], to R
work
and R
free
of 18.7% and 22.4%, respectively (Table 1). No
noncrystallographic symmetry constraints were used, but
a TLS model was applied in the last refinement runs in
refmac, with one TLS group for each domain of EF-G.
The structure contains two molecules in the asymmetric
unit, resulting in 51.1% solvent content.
Structure analysis
Calculations of radii of gyration were performed with
moleman2 [46] from the corresponding coordinates depos-
ited in the PDB. Structure superposition was performed
with o [47].
Acknowledgements
We thank the SLS and ESRF for beam time and sup-
port during data collection, and K. Ba
¨
ckbro 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
Carl Trygger’s Foundation to M. Selmer and S. San-

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