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 structure
Trang 1analysis 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 facfac-tors, in prokaryotes
initia-tion factor 2, elongainitia-tion factor Tu (EF-Tu), elongainitia-tion
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 elongaelonga-tion, 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 aureusto 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 thermophilusEF-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.
Trang 2binds 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 P21 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 AV 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
Refinement statistics
Number of atoms
Rmsd from ideality
Ramachandran statistics
a
Values in parentheses represent the highest-resolution bin.
Trang 3indicated 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 aureusEF-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 Caatoms), 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
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.
Trang 4T thermophilusEF-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 AGso 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
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).
Trang 5C
B
D
E
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
region of S aureus EF-G contoured at 3r Omitted residues are shown in yellow stick representation (D) Comparison of the switch II region
thermo-philus EF-G wild type (PDB 1elo, yellow [13]); T thermothermo-philus EF-G H573A (PDB 1fnm, orange [16]); T thermothermo-philus 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
residues 407–474), showing how the FA-binding site in the S aureus EF-G structure is blocked by the switch II region.
Trang 6arrangement, 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 BGand Tyr126 in helix
CG 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
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
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.
Trang 7a 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-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 AIII
packs against the central part of helix BIII (Fig 4B) Asp434 and Thr436 in helix AIII both form hydrogen bonds with His457 in helix BIII Thus, the mutations P435Q, T436I, H457Y and P435Q will change this
Trang 85G
5G
CG
CG
CG
CG
DG
CG
Trang 9AIII
Leu Arg Gln
AIII
AIII
AIII
BIII
AIII
AIII
BIII
AIII
Ser Cys Val
BIII
BIII
Trang 10BIII
BIII
BIII
BIII
Ser His Leu Cys
BIII
AIII
AV
AV
2V
3V
BV
BV