Download Mycobacterium tuberculosis DNA gyrase ATPase domain structures

Biochem. J. (2013) 456, 263–273 (Printed in Great Britain)
doi:10.1042/BJ20130538
263
Mycobacterium tuberculosis DNA gyrase ATPase domain structures suggest
a dissociative mechanism that explains how ATP hydrolysis is coupled to
domain motion
Alka AGRAWAL*1 , Mélanie ROUɆ‡§1 , Claus SPITZFADEN*, Stéphanie PETRELLA†‡§, Alexandra AUBRY¶**, Michael HANN*,
Benjamin BAX*2 and Claudine MAYER†‡§
*Platform Technology Sciences, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K., †Unité de Microbiologie Structurale,
Institut Pasteur, 75015 Paris, France, ‡URA 2185, CNRS, 75015 Paris, France, §Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, 75015 Paris, France, UPMC Université
Paris 06, ER5, EA 1541, Laboratoire de Bactériologie-Hygiène, Paris, France, ¶AP-HP, Hôpital Pitié-Salpêtrière, Laboratoire de Bactériologie-Hygiène, Paris, France, and **Centre
National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Paris, France
DNA gyrase, a type II topoisomerase, regulates DNA topology
by creating a double-stranded break in one DNA duplex and
transporting another DNA duplex [T-DNA (transported DNA)]
through this break. The ATPase domains dimerize, in the
presence of ATP, to trap the T-DNA segment. Hydrolysis of only
one of the two ATPs, and release of the resulting Pi , is ratelimiting in DNA strand passage. A long unresolved puzzle is
how the non-hydrolysable ATP analogue AMP-PNP (adenosine
5 -[β,γ -imido]triphosphate) can catalyse one round of DNA
strand passage without Pi release. In the present paper we
discuss two crystal structures of the Mycobacterium tuberculosis
DNA gyrase ATPase domain: one complexed with AMP-PCP
(adenosine 5 -[β,γ -methylene]triphosphate) was unexpectedly
monomeric, the other, an AMP-PNP complex, crystallized as
a dimer. In the AMP-PNP structure, the unprotonated nitrogen
(P-N = P imino) accepts hydrogen bonds from a well-ordered
‘ATP lid’, which is known to be required for dimerization. The
equivalent CH2 group, in AMP-PCP, cannot accept hydrogen
bonds, leaving the ‘ATP lid’ region disordered. Further analysis
suggested that AMP-PNP can be converted from the imino
(P-N = P) form into the imido form (P-NH-P) during the catalytic
cycle. A main-chain NH is proposed to move to either protonate
AMP-P-N = P to AMP-P-NH-P, or to protonate ATP to initiate
ATP hydrolysis. This suggests a novel dissociative mechanism
for ATP hydrolysis that could be applicable not only to GHKL
phosphotransferases, but also to unrelated ATPases and GTPases
such as Ras. On the basis of the domain orientation in our
AMP-PCP structure we propose a mechanochemical scheme to
explain how ATP hydrolysis is coupled to domain motion.
INTRODUCTION
together [6] in a way that stimulates both ATP-gate dimerization
[7,8] and ATPase activity [9,10].
The structure of the ATPase domain of the GyrB subunit of
Escherichia coli DNA gyrase [7] showed that it contained two
structural domains, an N-terminal GHKL domain [11] and a Cterminal transducer domain (Figure 1). A change in the relative
positions of the GHKL and transducer domains in response
to the hydrolysis of the first ATP [12,13] is important in guiding
the T-DNA segment through the cleaved DNA-gate during the
catalytic cycle [1]. The structures of eukaryotic [12,14], bacterial
[7,8,15] and archaeal [13,16] ATPase domains in complex with
the non-hydrolysable ATP analogue, AMP-PNP (adenosine 5 [β,γ -imido]triphosphate), are dimeric in the crystal, with the
GHKL and transducer sub-domains of each monomer held in an
‘ATP-restrained’ conformation [13]. In contrast, the complexes
with inhibitors or the apo forms are in an ‘open’ or ‘relaxed’
conformation [13,16,18]. The GHKL domain is named after DNA
gyrase, HSP90 (heat-shock protein of 90 kDa), histidine kinases
and MutL, four proteins all found to possess domains with the
same ATP-binding fold [11]. Three of these, known as the GHL
ATPases, transfer the terminal phosphate to a water (or OH − ion),
Type II DNA topoisomerases are essential nucleic acid-dependent
nanomachines present in all organisms. They solve topological
problems of DNA by temporarily introducing a double-stranded
break into one DNA segment and transporting another DNA [TDNA (transported DNA)] duplex through this temporary break
[1,2]. Most bacteria have two type II topoisomerases (DNA gyrase
and Topo IV), which are are heterotetrameric enzymes composed
of two subunits (A2 B2 ). However, the Mycobacterium tuberculosis
genome posseses only one type II topoisomerase, DNA gyrase.
M. tuberculosis DNA gyrase is capable of introducing negative
supercoils into DNA and has decatenation activity [3]. The
structures of all of the M. tuberculosis GyrA domains and the
C-terminal domain of M. tuberculosis GyrB have previously been
determined [4,5]. In the present study, the focus is on the structure
of the N-terminal ATPase domain of M. tuberculosis GyrB.
Type II topoisomerases function as ATP-dependent clamps
(Figure 1). The ATP-gate closes to capture the T-DNA segment
and remains closed when the central G-DNA (gate DNA) opens.
Capture of a T-DNA segment brings the ATPase domains closer
Key words: ATPase domain, ATP hydrolysis, dissociative mechanism, DNA gyrase.
Abbreviations used: AMP-PCP, adenosine 5 -[β,γ-methylene]triphosphate; AMP-PNP, adenosine 5 -[β,γ-imido]triphosphate; ASEC, analytical sizeexclusion chromatography; AUC, analytical ultracentrifugation; CSD, Cambridge Structural Database; HSP90, heat-shock protein of 90 kDa; P-loop,
phosphate-binding loop; T-DNA, transported DNA.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed (email [email protected]).
Co-ordinates and structure factor files for the M. tuberculosis GyrB ATPase domain have been deposited in the PDB under the accession codes 3ZKB,
3ZKD and 3ZM7.
c The Authors Journal compilation c 2013 Biochemical Society
264
Figure 1
A. Agrawal and others
Domains in the GyrB subunit of M. tuberculosis DNA gyrase and activity of the ATPase domain
(A) M. tuberculosis DNA gyrase consists of two subunits GyrA and GyrB. The ATPase domain of GyrB (residues 1–427) contains two structural domains: a GHKL domain (residues 21–255) and
a transducer domain (residues 256–427). (B) Simplified schematic diagram illustrating how the ATP-gate closes to capture the T-DNA segment (DNA, black cylinders) and remains closed when
the central DNA-gate opens. GyrA subunits are white and structural domains in GyrB subunits are coloured as in (A). (C) ATPase activity of the ATPase domain (Mtb GyrB47) was assessed (nM of
phosphate produced per s) at room temperature for 30 min at 15 μM protein and various ATP concentrations.
and also have the transducer domain and a residue equivalent to
Glu42 in E. coli GyrB [11]. Jackson and Maxwell [19] identified
Glu42 as a key catalytic residue in the ATPase reaction of DNA
gyrase and proposed that it acts as a general base polarizing a
water molecule for nucleophilic attack on the γ -phosphate. In
a comprehensive paper, Corbett and Berger [13] solved structures
with several ADP and ATP analogues to structurally dissect
ATP turnover in the prototypical GHL ATPase TopoVI. This
led to the proposal of a detailed mechanism [13] for ATP
hydrolysis (Supplementary Figure S1A at http://www.biochemj.
org/bj/456/bj4560263add.htm), which is initiated when the
conserved glutamate residue (equivalent to Glu42 in E. coli
GyrB) abstracts a proton from a water (consistent with the
original proposal of Jackson and Maxwell [19]). In contrast, in a
dissociative mechanism [20], the breaking of the bond between the
β- and γ -phosphates (Supplementary Figure S1B), gives a highly
electrophilic metaphosphate [PO3 ] − ion and a protonated ADP2 −
ion (Supplementary Figure S2 at http://www.biochemj.org/bj/
456/bj4560263add.htm).
In the present paper, we report crystal structures of the
M. tuberculosis GyrB ATPase domain (MtbGyrB47) with
two different non-hydrolysable ATP analogues; purification,
crystallization and data collection are reported elsewhere [21].
A structure with AMP-PCP (adenosine 5 -[β,γ -methylene]
triphosphate) is the first non-dimeric structure of a type II
topoisomerase ATPase domain with an ATP analogue, whereas
two AMP-PNP structures are dimeric and similar to previously
determined ‘ATP-restrained’ structures. This gives new insights
into the catalytic cycle of M. tuberculosis DNA gyrase, and
provides a structural explanation of why ‘non-hydrolysable’
AMP-PNP can drive one round of strand passage in type II
topoisomerases [22,23], whereas, with ATP, the release of Pi from
the hydrolysed ATP is the rate-limiting step [24,25]. We note
that whereas the bridging nitrogen of AMP-PNP (Supplementary
Figure S2) is normally protonated (P-NH-P, imido), in the
presence of divalent metal ions, it is often unprotonated (P-N = P,
imino) [26].
MATERIALS AND METHODS
line, U.K. and Pasteur Institute, France respectively). Both constructs coded for residues 1–427 of M. tuberculosis GyrB, but they
had slightly different N-terminal His6 tags. For MtbGyrB47C1 ,
the His6 tag was systematically cleaved. For MtbGyrB47C2 , the
protein was purified with the His6 tag intact. The N-terminal tags
were not seen in crystal structures and made, within experimental
error, no significant difference in ATPase activity assays. Details
of the expression, purification, crystallization and data collection
on crystals of MtbGyrB47C1 with AMP-PNP and of MtbGyrB47C2
with AMP-PCP are reported elsewhere [21].
ATP hydrolysis assays
ATPase activity of the M. tuberculosis DNA gyrase was assessed
at the Pasteur Institute by measurement of free Pi using the
pyruvate kinase/lactate dehydrogenase assay described previously
[27]. The reaction mixture (100 μl) contained 50 mM Tris/HCl
(pH 7.5), 50 mM KCl, 5 mM MgCl2 , 0.25 mM NADH, 1 mM
phosphoenolpyruvate, 2 units of pyruvate kinase and 2 units
of lactate dehydrogenase,various amounts of ATP and M.
tuberculosis DNA gyrase [an equimolar mixture of GyrA and
GyrB subunits in 50 mM Tris/HCl (pH 8) and 50 mM NaCl].
Reactions were performed in the absence or presence of DNA
(relaxed pBR322, 5 μg/μl) at 37 ◦ C and the decrease in NADH
concentration was monitored continuously as a function of time
for 90 min by measuring the absorbance at 340 nm in a UV–visible
spectrophotometer. The K m and V max values were determined from
the double-reciprocal plots.
ATPase activities of the ATPase domain and GyrB subunit of the
M. tuberculosis DNA gyrase were assessed at GlaxoSmithKline
by measurement of free Pi using the fluorescence method
described in [28]. The proteins were first buffer-exchanged
into 20 mM Tris (pH 7.5), 100 mM NaCl and 1 mM EDTA to
remove DTT, as this can produce erroneous results. Assays were
carried out in 10 μl aliquots in triplicate, and reactions were
followed for 30 min at room temperature (22 ◦ C). Fluorescence
was measured using a Gemini microplate spectrofluorometer
and Softmax Pro (Molecular Devices). Initial rates were correlated with a phosphate calibration curve and kinetics were
analysed with Grafit (Erithacus Software).
Protein expression, purification, crystallization and data collection
Two constructs, MtbGyrB47C1 and MtbGyrB47C2 , for the ATPase
domain of M. tuberculosis H37Rv GyrB (residues 1–427) were designed independently in two different laboratories (GlaxoSmithK
c The Authors Journal compilation c 2013 Biochemical Society
Structure determination and refinement
The structure of the P1(8) crystal form of MtbGyrB47C1
with AMP-PNP was determined by molecular replacement
ATP hydrolysis by the M. tuberculosis DNA gyrase ATPase domain
Table 1
265
Refinement parameters
Values in parentheses correspond to the highest-resolution outer shell. Data collection parameters are in [21].
Parameter
AMP-PNP
AMP-PNP
AMP-PCP
Space group
Number of subunits in the asymmetric unit
Resolution (Å)
Resolution range (Å)
Completeness (%)
Number of reflections
R work /R free
Number of atoms
Protein
Heteroatoms
Solvent
Mean B value (Å2 )
RMSDs
Bond length (Å)
Bond angles (◦ )
Ramachandran plot
Favoured (%)
Allowed (%)
Disallowed (%)
P1
8
40–2.95 (3.0–2.95)
25–2.95
84.6
61070
18.2/25.7
P1
16
25–2.9 (2.95–2.9)
25–2.90
98.2
159159
18.2/24.0
P 21
6
50–3.3 (3.5–3.3)
25–3.3
99.7
40735
19.6/22.3
22821
371
115
80.3
45997
955
439
99.1
16012
283
91
113.9
0.009
1.16
0.009
1.10
0.010
1.19
87.9
11.9
0.2
90.3
9.1
0.1
90.2
9.8
0.0
with PHASER [29] using the 43 kDa E. coli ATPase domain
(40 % amino acid identity) as the search model (PDB code 1EI1)
[8] and refined. The P1(16) crystal form of MtbGyrB47C1 (with
AMP-PNP) was solved by molecular replacement from the P1(8)
crystal form and refined (statistics in Table 1). In the final models
of both of the AMP-PNP crystal forms the bridging nitrogen
has been modelled in the imino form (P-N = P). Refinement
of the AMP-PNP structures was carried out with Refmac [30],
phenix.refine [31] and Buster [32]. Restraint dictionaries for
the imino (P-N = P) form of AMP-PNP were generated with
eLBOW [33] and fit [34] into difference maps calculated
with phenix.refine [31]. In the final 2.9 Å (1 Å = 0.1 nm) AMPPNP structure the main-chain NH groups of Leu120 and Gly122
are 3.09 and 3.12Å from the bridging nitrogen (quoted distances
are averages of sixteen observations for each in the asymmetric
unit; errors on distances estimated to be ∼ 0.1 Å). Analysis of
a closely related higher-resolution structure with AMP-PNP, a
1.87 Å human Topo II structure (PDB code 1ZXM), confirmed
that the bridging nitrogen between the β- and γ -phosphate
AMP-PNP could not be protonated (Supplementary Figure S3
at http://www.biochemj.org/bj/456/bj4560263add.htm). Small
molecule crystal structures in the CSD (Cambridge Structural
Database) [35], which have a bridging nitrogen between two
phosphate groups, were analysed with Conquest, Mercury and
Mogul [36]. This analysis suggested that if two oxygens from
the two phosphate groups co-ordinate a divalent metal ion,
then the bridging nitrogen of AMP-PNP was likely to be in the
imino (P-N = P or P = N-P) form (Supplementary Figure S2). In
this unprotonated form the two P-N bond lengths are nearly equal
in length (1.59 Å), suggesting delocalized π-bonding. In smallmolecule crystal structures in the CSD in which the nitrogen
between the two phosphates was protonated (P-NH-P, imido; see
Supplementary Figure S2), the P-N bond lengths were longer
◦
(1.64 Å), the P-N-P bond angle was 130 +
− 1 (compared with
◦
123 +
3
in
the
unprotonated
structures)
and
co-ordination
of a
−
divalent metal ion was not observed. In small-molecule crystal
structures and in protein complexes with AMP-PNP, the presence
of a divalent metal ion tends to bring the two co-ordinating
oxygens from the two phosphates quite close together (<3 Å),
so that looking along the virtual P-P bond, the two phosphate
groups are nearly eclipsed (Supplementary Figure S3). In P-NH-P
structures looking along the virtual P-P bond the two phosphates
tend to be staggered (results not shown), presumably because
of electrostatic repulsion between negatively charged phosphate
oxygens. Whereas in both the imido (P-NH-P) and imino (PN = P) forms of AMP-PNP the nitrogen appears largely SP2 in
character, analysis of small-molecule crystal structures suggests
that in AMP-PCP the CH2 group is SP3 in character, with P-C
◦
bond distances of 1.80 Å and a P-C-P bond angle of 115 +
−3 .
C2
The structure of MtbGyrB47 with AMP-PCP was determined
by molecular replacement with PHASER [29] using GHKL and
transducer domains from the refined MtbGyrB47–AMP-PNP as
the search models. Structure refinement was carried out with
BUSTER [32] to 3.3 Å. Model building was performed with
Coot [37]. Model refinement statistics are summarized in Table 1
and structure factors and co-ordinates have been deposited in the
PDB under codes 3ZKB (2.9 Å, AMP-PNP), 3ZKD (2.95 Å,
AMP-PNP) and 3ZM7 (3.3 Å, AMP-PCP). Structural Figures
were drawn with PyMOL (http://www.pymol.org) unless stated
otherwise.
AUC (analytical ultracentrifugation) and analytical ASEC
(analytical size-exclusion chromatograohy)
Sedimentation velocity experiments were performed at 15 μM
protein (MtbGyrB47C2 or His6 –MtbGyrB47C2 ) either in Tris buffer
[50 mM Tris (pH 8) and 50 mM NaCl, +
− 5 mM AMP-PCP or
5 mM AMP-PNP] or in PBS (pH 7.4, +
− 2 mM AMP-PNP and
+
− 2 mM Mg or 2 mM EDTA). The PBS samples were gently
resuspended at the end of the AUC run and the run repeated
with the same sample cell after 3 or 6 days of incubation at room
temperature.
Experiments were performed in a Beckman XL-I analytical
ultracentrifuge using a double sector charcoal-Epon cell at 20 ◦ C
and 42 000 rev./min. Interference scans were taken every 6 min.
The program Sednterp 1.09 (available at http://www.jphilo/
mailway.com/download.htm) was used to calculate solvent
density, solvent viscosity and partial specific volume using the
amino-acid composition. The sedimentation data were analysed
c The Authors Journal compilation c 2013 Biochemical Society
266
A. Agrawal and others
with the program Sedfit [39] using the continuous c(s) and
c(M) distributions. The theoretical sedimentation coefficient value
calculated from the crystallographic monomer structure without
the ATP lid and insertion region (see below) was 3.8 S. AUC
experiments showed that the MtbGyrB47C2 behaved as a monomer
in the absence or presence of AMP-PCP, irrespective of the
presence or absence of the N-terminal His6 tag.
The oligomerization state of MtbGyrB47C1 and MtbGyrB47C2
in solution was also assessed by ASEC. MtbGyrB47C1 was mixed
with 5 mM MgCl2 plus 1 mM AMP-PNP (Sigma) and applied to a
TOSOH SW3000 column in 20 mM Hepes (pH 7.5) and 100 mM
Na2 SO4 at 0.2 ml/min. Cytochrome c (12.4 kDa), carbonic
anhydrase (29 kDa), BSA (66 kDa), alcohol dehydrogenase
(150 kDa), β-amylase (200 kDa), apoferritin (443 kDa) and
thyroglobulin (669 kDa) from Sigma were used to calibrate the
column.
ASEC showed slow time-dependent dimerization on a time
scale of days in the presence of AMP-PNP (results not shown),
in agreement with the results from AUC. Dimerization was not
observed in the presence of novobiocin, consistent with results
for the E. coli GyrB ATPase domain [27].
RESULTS
ATPase activity of M. tuberculosis DNA gyrase
ATPase activities of full-length M. tuberculosis GyrB and
the ATPase domain (MtbGyrB47, residues 1–427) measured
independently in two laboratories with different assays (see the
Materials and methods section) gave similar results.
To confirm that purified MtbGyrB47 was active, we analysed
its ATPase activity using a sensitive fluorescence assay which
measures the production of Pi [28]. As the ATPase activity of
the isolated ATPase domain is quite low, 15 μM protein was
used. Figure 1 shows an ATP titration for the ATPase domain.
An ATP-dependent increase in activity was observed, and activity
was Mg2 + -dependent and inhibited by novobiocin (results not
shown). The kcat was 0.002 s − 1 . Full-length GyrB is also active,
with a kcat of 0.025 s − 1 at 15-fold less protein (results not shown),
suggesting that the C-terminal end of the protein (missing from
the isolated ATPase domain) enhances ATPase activity, possibly
by making M. tuberculosis GyrB dimeric. This higher activity
of the full-length protein compared with the ATPase domain has
also been observed in Saccharomyces cerevisiae and Plasmodium
falciparum Topo II [40,41].
The ATPase activity of M. tuberculosis DNA gyrase
(GyrA and GyrB subunits at an equimolar concentration)
was investigated using a PK/LDH (pyruvate kinase/lactate
dehydrogenase)-linked enzyme assay as a function of enzyme
concentration at a fixed substrate concentration. The ATPase
activity of the M. tuberculosis DNA gyrase shows a linear
dependence of the rate of hydrolysis depending on the enzyme
concentration (Supplementary Figure S4 at http://www.biochemj.
org/bj/456/bj4560263add.htm). Typically, 5 μM of the M.
tuberculosis gyrase was found to have an activity of 30 nM/s,
and activity was enhanced at least 1.5-fold (from 1.5 to 2.5) in
the presence of DNA (Supplementary Figure S4). The ATPase
activity of the M. tuberculosis gyrase demonstrated a hyperbolic
dependence on substrate concentration with a K m (app) of
0.77 mM and a kcat (app), or enzyme turnover number, of
0.02 s − 1 (Supplementary Figure S4). In comparison with the
values observed for the E. coli DNA gyrase (K m = 0.83 mM and
kcat = 1.2 s − 1 ) [10], the K m (app) is similar, but the kcat (app) was
decreased by 60-fold, resulting in a decreased catalytic efficacy
for the M. tuberculosis gyrase. The much lower ATPase activity
c The Authors Journal compilation c 2013 Biochemical Society
of M. tuberculosis gyrase compared with E. coli gyrase has also
recently been reported elsewhere [5].
The structures of the M. tuberculosis ATPase domain in complex
with AMP-PCP and AMP-PNP
Structures of the M. tuberculosis ATPase domain with AMP-PCP
and AMP-PNP were determined by molecular replacement and
refined as described in the Materials and methods section (see
Table 1 for details). The structure of MtbGyrB47 with AMP-PCP
was, surprisingly, not dimeric (Figures 2A–2C). There was clear
density for the AMP-PCP in all six subunits in the asymmetric unit
(Figure 2C and Supplementary Figure S5 at http://www.biochemj.
org/bj/456/bj4560263add.htm), but the ATP lid region (residues
104–125) was largely disordered, and the GHKL and transducer
domains were differently oriented than in the AMP-PNP structure
(see the next section for more detail).
The structure of MtbGyrB47 with AMP-PNP was dimeric
(Figures 2D and 2E), had an ordered ATP lid region and the
dimer was stabilized by the N-terminal arm crossing from one
subunit to the other. The overall dimeric structure of MtbGyrB47
with AMP-PNP is similar to previously reported AMP-PNP
complexes of ATPase domains (Supplementary Figure S6 at http://
www.biochemj.org/bj/456/bj4560263add.htm) from E. coli GyrB
[7,8], E. coli Topo IV [15], human Topo II [12] and other type II
topoisomerases. Two main-chain NHs were within hydrogenbonding distance of the bridging nitrogen in the AMP-PNP
structure (Figure 2F) showing it to be in the unprotonated (PN = P) imino form [26]. In the MtbGyrB47–AMP-PNP structures
the ordered ATP lid region (residues 104–124) wraps around the
γ -phosphate and also makes extensive interactions with the Nterminal arm from the other subunit of the dimer (Figures 2D–
2F). Whereas most of the contacts to the AMP-PNP come from
the GHKL domain (residues 21–255), Lys372 from the transducer
domain (Lys337 in E. coli) contacts the γ -phosphate of ATP, as
observed in related structures. In the AMP-PCP structure this
switch lysine residue does not contact the γ -phosphate.
Compared with previously determined crystal structures
of bacterial type II topoisomerase ATPase domains, the M.
tuberculosis GyrB ATPase domain possesses an insert of 32
amino acids between the last two β-strands of the GHKL
domain (Supplementary Figure S7 at http://www.biochemj.org/
bj/456/bj4560263add.htm). In our MtbGyrB47 structures this
insert (residues 214–245) is largely disordered (Figures 2A and
2D). PSI-Blast searches showed that this insert (residues 214–
245) is found only in bacterial GyrB ATPase domains of the
Gram-positive Corynebacterineae, which include Mycobacteria,
Nocardia, Rhodococcus, Gordonia and Corynebacteria [42].
In the MtbGyrB47–AMP-PCP structure there were six subunits in
the asymmetric unit, all structurally similar, arranged in two very
similar trimers. The insert region (residues 214–245) was close
to the three-fold axis of the trimers (Supplementary Figure S8
at http://www.biochemj.org/bj/456/bj4560263add.htm); however,
there was not clear electron density for the insert in the 3.3 Å maps,
so the insert was not modelled. AUC experiments of MtbGyrB47
with AMP-PCP showed monomers in solution (Supplementary
Figure S9 at http://www.biochemj.org/bj/456/bj4560263add.
htm), so the trimers seen in the crystal structure are probably not
biologically relevant. The two crystal forms solved with AMPPNP had either four dimers or eight dimers in the asymmetric
unit and, in one subunit of each dimer, a short β-strand at the
end of the insert region (residues 243–246) mediated a common
crystal contact (Supplementary Figure S8C). In one of the 24
AMP-PNP subunits a longer ordered region was stabilized by
ATP hydrolysis by the M. tuberculosis DNA gyrase ATPase domain
Figure 2
267
Structures of the M. tuberculosis ATPase domain with AMP-PCP and AMP-PNP
(A and B) Two orthogonal views of the monomeric structure of Mtb GyrB47 with AMP-PCP. AMP-PCP, red sticks; Mg2 + blue sphere. The ATP lid (residues 104–125, green) is largely disordered. (C)
F o − F c (3 σ ) omit map (mesh) for the AMP-PCP (carbon, magenta; nitrogen, blue; oxygen, red; and phosphate, orange) and Mg2 + (small blue sphere). The protein is shown with yellow carbons,
except for residues in the ATP lid which have green carbons (residues 104 and 122–125 are included in the model, residues 105–121 are disordered). (D and E) Two orthogonal views of the dimeric
structure of Mtb GyrB47 with AMP-PNP. AMP-PNP, red sticks; Mg2 + , blue sphere. The ATP lid (residues 104–125, green), which is shown as a solid main-chain trace and semi-transparent spheres
(green) for all atoms, buries the AMP-PNP and interacts with the N-terminal arm (black) from the other subunit of the dimer. (F) F o − F c (3 σ ) omit map (mesh) for AMP-PNP and Mg2 + [coloured
as in (C), except that protein atoms are with brown carbon atoms when not in the ATP lid]. The black broken lines indicate that the main-chain NH groups of Leu120 and Gly122 are 3.09 and 3.12 Å
from the bridging nitrogen. (G) Superimposition of the three conformations: RelaxT, observed in the AMP-PCP structure (yellow); ATS, ATP-restrained (AMP-PNP) conformation (orange); RelaxED,
relaxed conformation (brown). For clarity, the AMP-PCP-bound structure was also used for the RelaxED structure, superimposing its GHKL and transducer domains on corresponding domains of
the T. thermophilus gyrase (RelaxED) structure. The red square and zoom-in view highlight different orientations of the C-terminal helix. (H) Comparison of the binding modes for AMP-PCP and
AMP-PNP. Note that the CH2 in AMP-PCP cannot accept hydrogen bonds from the NH groups of Leu120 and Gly122 .
a unique crystal contact. A sequence search of structures in
the PDB revealed that the N-terminal domain of HSP90 from
P. falciparum also possesses a similar, but longer, insert (50 %
sequence similarity over the 32 residues in common), also located
inbetween the last two strands of a GHKL domain (PDB code
3PEH) (Supplementary Figure S8B). However, no function was
suggested for this insert in P. falciparum HSP90 [43], and although
our structural studies have not identified a clear functional role for
the M. tuberculosis GyrB insert, crystal packing contacts suggest
it may play a role in protein–protein interactions.
Comparison of the M. tuberculosis ATPase domain with AMP-PCP
and AMP-PNP
The MtbGyrB47 structure with AMP-PCP is the first nondimeric structure of a DNA gyrase ATPase domain in complex
with an ATP analogue. Comparing the AMP-PCP subunit
structure to that with AMP-PNP (Figure 2G), the individual
GHKL and transducer subdomains are similar (Cα RMSD of
0.3 Å over 152 atoms for GHKL, and Cα RMSD of 0.3 Å
over 136 atoms for transducer), but the relative orientation of
c The Authors Journal compilation c 2013 Biochemical Society
268
A. Agrawal and others
for binding and catalysis are observed in M. tuberculosis gyrase.
In the AMP-PNP structures the ATP lid is ordered. Residues at the
C-terminus of the ATP lid, GLHGVG (residues 119–124), form
a glycine-rich P-loop (phosphate-binding loop), that has mainchain NH groups from His121 , Val123 and Gly124 , making hydrogen
bonds to oxygens on the γ -phosphate of AMP-PNP. Gly122 has its
main-chain NH pointing directly at the bridging nitrogen between
the β- and γ -phosphates, whereas the main-chain nitrogen
of Leu120 is within 3.0 Å of both the bridging nitrogen and one of
the oxygens on the γ -phosphate (Figure 4A). The binding mode
observed for AMP-PNP in MtbGyrB47 is essentially the same
as seen in the 1.8 Å yeast Topo II complex (Figure 4B) and the
1.87 Å human complex (Supplementary Figure S3); we conclude
that these structures also bind the imino form of AMP-PNP. A
more distantly related Topo VIB structure with AMP-PNP [16] is
also in the imino (P-N = P) form [26].
Two E. coli Topo II structures appear to contain the imido (P-NH-P)
form of AMP-PNP
Figure 3
AUC of the M. tuberculosis GyrB ATPase domain
AUC traces are shown of untagged Mtb GyrB47C2 at 17 μM (bottom) and Mtb GyrBC2 in the
presence of 2 mM MgCl2 and 2 mM AMP-PNP (top). Time points shown are for the fresh
samples (), after incubation at room temperature for 3 days ( ) and
6 days (). MWapp, apparent molecullar mass.
the GHKL and transducer subdomains are different, with a
swivel of approximately 17◦ , moving the distal end of the Cterminal-most helix approximately 15 Å outward (Figure 2G).
This movement is different from the one observed for the
relaxed conformation of type II topoisomerase ATPase domains
(Figure 2G). The MtbGyrB47–AMP-PCP structure seems to be in
a new conformational state, which we term RelaxT (Figure 2G),
intermediate between the previously observed RelaxED (or open)
and ATP-restrained (ATS or closed) conformations [13,18]. In
the AMP-PCP structure the ATP lid region (residues 104–125)
is disordered, as it is in many structures with inhibitors [44] and
in some structures with ADP [13]. This suggested that because
the CH2 between the β- and γ -phosphates of AMP-PCP cannot
accept hydrogen bonds from main-chain NHs of Leu120 and Gly122
(Figures 2C, 2F and 2H), the ATP lid cannot become ordered and
close, and therefore the dimer cannot form.
AUC showed that incubation of the M. tuberculosis GyrB
ATPase domain with AMP-PNP initially gave monomers (as
observed with AMP-PCP and apo protein), but that dimers formed
slowly (compared with the 6-10 h run time of an AUC experiment)
over a period of several days (Figure 3). This suggested that
AMP-PNP was initially largely in the imido (P-NH-P) form, and
that the presence of the hydrogen on the nitrogen blocked the
ATP lid from becoming ordered and closing (as with AMP-PCP).
However, the AMP-PNP could convert into the imino (P-N = P)
form, and when in the imino (P-N = P) form the ATP lid could
close and the dimer form; the percentage of dimer increased with
time (Figure 3). Crystals of MtbGyrB47 with AMP-PNP were
grown at pH 8.5, in the presence of between 5 and 200 mM MgCl2
[21], conditions expected to stabilize the imino (P-N = P) form.
This slow interaction of AMP-PNP has also been observed with
E. coli DNA gyrase [45].
Several Topo II crystal structures contain the imino (P-N = P) form
of AMP-PNP
The ATP-binding pocket of type II topoisomerase ATPase
domains is highly conserved and all of the important residues
c The Authors Journal compilation c 2013 Biochemical Society
The position of the nitrogen between the β- and γ -phosphates in
a 2.3 Å E. coli gyrase and a 2.1 Å E. coli Topo IV structure
with AMP-PNP are quite similar to each other (Figures 4C
and 4D), but this nitrogen is in a different position from other
structures (Figure 4). Moreover, in these two structures there is
not a Mg2 + adjacent to the β- or γ -phosphates (although the
2.1 Å E. coli Topo IV structure has a nearby Mg2 + ; Figure 4D).
The different position of the bridging nitrogen (and the absence
of the adjacent Mg2 + ion) in the E. coli gyrase and Topo IV
structures (Figures 4C–4E) suggests that the nitrogen is in the
imido form (Supplementary Figure S2). In the 2.1 Å E. coli Topo
IV structure (Figure 4D), the closest main-chain NH from the
ATP lid is some 3.9 Å away; the bridging nitrogen is too far away
to accept hydrogen bonds from the ATP lid. However, the ATP lid
is ordered and in the same conformation as observed in the other
AMP-PNP structures, moreover these two E. coli structures are
dimeric (Supplementary Figure S6). This observation was initially
puzzling because the MtbGyrB47 AMP-PCP structure suggested
that to initially become ordered and close, the ATP lid needed to
make contact with the atom between the β- and γ -phosphates.
Only once the ATP lid has closed can the dimer form.
One possibility is that dimers of the two E. coli proteins formed
while the AMP-PNP was in the imino form (P-N = P), but at
some point during crystal formation the NH group of the adjacent
glycine residue protonated the P-N = P group to be P-NH-P.
In the scheme shown in Figure 5 (Figures 5A–5C), movement
of the P-loop and the AMP-PNP would then allow a hydrogen
from the γ -phosphate to reprotonate the glycine residue. The
extensive interactions with the N-terminal arm of the other subunit
(Figures 2D and 2E) would keep the ATP lid ordered after the
AMP-PNP had adopted the imido form.
DISCUSSION
A fully dissociative mechanism for ATP hydrolysis
The proposed scheme for the catalytic conversion of the imino (PN = P) into the imido (P-NH-P) form of AMP-PNP (Figures 5A–
5C) suggests a simple dissociative mechanism for ATP hydrolysis
by M. tuberculosis GyrB and related ATPases (Figures 5G–5I).
In this proposed mechanism an initial movement of Gly122 causes
its main-chain NH to protonate the bridging oxygen of the ATP.
Stabilization of the resulting negative charge on the protein is
proposed to be, in M. tuberculosis gyrase, by transfer to Tyr33
ATP hydrolysis by the M. tuberculosis DNA gyrase ATPase domain
Figure 4
269
In structures of AMP-PNP with type IIA topoisomerases, the nitrogen between the β- and γ -phosphates is found in two different positions
(A) The 2.9 Å structure of M. tuberculosis GyrB (the present study). (B) The 1.8 Å structure of S. cerevisiae (PDB code 1PVG). (C) The 2.3 Å structure of E. coli GyrB (PDB code 1EI1). (D) The 2.1 Å
structure of E. coli Topo IV (PDB code 1S16). (E) Structures in (A–D) are superimposed. The two orientations for the bridging nitrogen are consistent with imino (P-N = P) or imido (P-NH-P) forms
of AMP-PNP.
via His104 (Figure 5D). In human Topo II a similar charge-relay
network (Figures 5E and 5F) involves His42 (equivalent to Ala25
in M. tuberculosis), although direct transfer of the charge to Tyr151
might also be possible. The protonation of ATP gives rise to an
ADP2 − and a free metaphosphate ion (Figure 5H). The activated
water immediately attacks the highly reactive metaphosphate ion
(Figure 5H) to give a phosphate ion. The ADP2 − (Supplementary
Figure S2) then reprotonates the main-chain nitrogen of Gly122
(Figure 5I), becoming an ADP3 − ion in the process. If the activated
water is not correctly positioned to attack the metaphosphate ion,
once the ADP3 − ion is formed it would be correctly positioned to
re-attack the metaphosphate ion to reform ATP [20].
A scheme for the reverse reaction, the synthesis of ATP by
type II topoisomerases [25], is shown in Supplementary Figure
S10 (at http://www.biochemj.org/bj/456/bj4560263add.htm). In
GHKL domain histidine kinases, the activated water is not present,
and the phosphate group is transferred from ATP to a histidine side
chain [46]. In two-component signal transduction the phosphate
group is then transferred from the histidine side chain to an
aspartic acid side chain (Supplementary Figure S10).
The proposed dissociative mechanism seems chemically
reasonable for GHKL domain histidine kinases. The previously
proposed mechanisms for ATP hydrolysis [13,19] by type II
topoisomerases show the reaction being initiated when the
activated water (or OH − ion) makes a nucleophilic attack on the
highly negatively charged γ -phosphate (Supplementary Figure
S1). This type of mechanism, initiated by a nucleophilic attack on
a negatively charged tetrahedral γ -phosphate, has been proposed
for many other ATPases and GTPases.
Could other ATPase/GTPase families also have a fully dissociated
mechanism?
The glycine-rich P-loop in M. tuberculosis GyrB and related
GHKL phosphotransferases does not correspond to the Walker
A motif (GxxxxGK[T/S]) found in many ATP- and GTP-binding
proteins [47]. However, because of structural similarities in the
way the atom corresponding to the bridging oxygen between the
β- and γ -phosphates is co-ordinated in transition state complexes
of Ras and F1 -ATPase and M. tuberculosis GyrB–AMPPNP (Supplementary Figure S11 at http://www.biochemj.org/
bj/456/bj4560263add.htm), it is tempting to speculate that Walker
A motif-containing proteins may have, by convergent evolution,
arrived at a similar mechanism for ATP/GTP hydrolysis. Main-
chain NHs from glycine residues point at the bridging oxygen
between the β- and γ -phosphates (Supplementary Figure S11) in
transition state analogue complexes of the small G-protein Ras
[48] (bold Gly13 in GaggvGKS) and in F1 -ATPase [49] (bold
Gly159 in GgagvGKT). We propose that movement of this glycine
residue past the bridging oxygen between the β- and γ -phosphates
forces it to leave its main-chain NH proton behind on the bridging
oxygen promoting GTP or ATP hydrolysis. The bridging oxygen
between the β- and γ -phosphates of GTP is negatively charged
[50]. A fully dissociative mechanism for GTP hydrolysis by
Ras (Supplementary Figure S12 at http://www.biochemj.org/bj/
456/bj4560263add.htm) can explain why point mutations at Gly12 ,
Gly13 or Gln61 impair intrinsic rates of GTP hydrolysis by
Ras, while increasing intrinsic rates of hydrolysis of some GTP
analogues [51].
How is ATP hydrolysis coupled to domain motion in M. tuberculosis
DNA gyrase?
Analysis of previously available type II topoismerase ATPase
structures (GHKL + transducer domains) supported the existence
of ‘ATP-restrained’ and ‘relaxED’ conformational states [13,18].
In the M. tuberculosis GyrB complex with AMP-PCP, a second
relaxed conformation (RelaxT) was observed, with the transducer
and GHKL domains in different relative positions; we term this
conformation ‘RelaxT’ and suggest it may correspond to the
monomeric ATP-bound form. In ‘ATP-restrained’ conformations
the size of the ‘hole’ between the two transducer domains is often
too small to accommodate the T-segment DNA [12,14].
In the present study we compare schemas showing how the
catalytic cycle of DNA gyrase could be governed by (i) ATP
hydrolysis [24,25] or (ii) protonation of imino AMP-P-N = P to
imido AMP-P-NH-P. The catalytic cycle in the presence of ATP
is described first (Figure 6A). A closed ATP lid is indicated by
a green square, an open ATP lid by a green line. When the ATP
lid is open, nucleotide exchange can take place. (i) At the start of
the catalytic cycle both GHKL domains are shown occupied by
ATP (represented by the letter T), but the two GHKL domains are
too far apart to dimerize. (ii) When the T-DNA comes between
the transducer domains, they are attracted inwards towards it
[6,52]. Once the two GHKL domains are close enough together
a closed ATP lid in one subunit will bind the terminal N-arm
from the other subunit, closing the ATP-gate dimer interface. (iii)
In forming this dimer the first ATPase domain adopts the ATS
c The Authors Journal compilation c 2013 Biochemical Society
270
Figure 5
A. Agrawal and others
Main-chain NH protonation of AMP-P-N = P to AMP-P-NH-P suggests a fully dissociative mechanism for ATP hydrolysis
(A–C) A scheme for enzymatic conversion of imino (P-N = P) into imido (P-NH-P) forms of AMP-PNP. (D) The Mtb GyrB47–AMP-PNP structure showing residues that could stabilize the negative
charge after Gly122 protonates ATP. (E) Human Topo II structure (PDB code 1ZXM) with AMP-PNP (equivalent view). (F) Superimposition of structures in (D) and (E). (G–I) A dissociative mechanism
for ATP hydrolysis by M. tuberculosis DNA gyrase.
conformation (orange) and the ATP lid hydrolyses the first ATP.
This first GHKL domain now contains ADP and Pi . (iv) When the
ATP lid in the second GHKL domain (which still contains ATP)
becomes ordered, the domain tries to adopt the ATP-restrained
conformation, but it cannot adopt this ATS conformation until
the T-DNA segment has been squeezed out from between the
two transducer domains. During this conformational change the
Pi is released from the first ATPase domain, allowing the first
ATPase domain, now containing ADP (D) to move to a relaxed
conformation. (v) The second ATPase domain can adopt the ATPrestrained conformation (orange) once the T-segment has passed
through the cleaved DNA, helping to close the central G-gate.
(vi) After the T-DNA segment has passed through the exit gate,
c The Authors Journal compilation c 2013 Biochemical Society
closure of the exit gate may signal for the second ATP to be
hydrolysed via interactions between DNA-gate domains and the
transducer domain [53]. In the presence of two ADPs (D), the
ATP lid is no longer restrained so tightly and the ATP-gate can
re-open, returning the enzyme to step (i) to rerun the catalytic
cycle.
The proposed schema when the catalytic cycle is regulated by
AMP-PNP (Figure 6B) is very similar to that for ATP. Note that
the ATP lid can only initially close when the AMP-PNP is in the
imino (P-N = P) form (shown by letter N in Figure 6B), but once
the dimer is formed and the ATP lid is held in place by extra
interactions with the N-arm of the other subunit, the ATP lid will
remain closed with the imido (P-NH-P) form (shown by letters NH
ATP hydrolysis by the M. tuberculosis DNA gyrase ATPase domain
Figure 6
271
Schemes illustrating coupling of ATP hydrolysis (or AMP-PNP protonation) with domain motion in M. tuberculosis gyrase
(A) ATP hydrolysis. T, ATP; D, ADP. (B) AMP-PNP protonation. NH, AMP-P-NH-P; N, AMP-P-N = P. Different conformations of the ATPase domains are indicated by different colours: yellow, RelaxT
conformation; orange, ATS conformation; brown, RelaxED conformation. A green central region in a GHKL domain indicates the ATP lid is closed. An open (disordered) ATP lid is indicated by a green
line. Disordered N-terminal arms (broken blue or black lines) become ordered (continuous blue or black lines) when they interact with an ordered ATP lid across the dimer interface. Gate DNA is
shown in black and T-DNA is in purple. (The C-terminal DNA-wrapping domain of GyrA is omitted for clarity). (C) Models of the ATPase domain of M. tuberculosis showing how the space between
the C-terminal helices increases between states (iii) and (iv) (as in A), allowing passage of the T (transport) segment DNA (purple). The RelaxED conformation (brown) was modelled on PDB code
1KIJ by superimposing the GHKL and transducer domains.
in Figure 6B). The protonation of the imino to the imido forms
is hypothesized to be accompanied by the expulsion of Mg2 +
normally co-ordinated by oxygens from β- and γ -phosphates. It
is suggested that the different conformation of the γ -phosphate
and the absence of Mg2 + allows the AMP-P-NH-P-bound ATPase
domain to adopt relaxed conformations.
Conclusions
The crystal structure of the M. tuberculosis DNA gyrase ATPase
domain with AMP-PCP was in a novel monomeric form, whereas
crystal structures with AMP-PNP were dimeric and similar to
many related structures in the literature. By including hydrogens
in the refinement and comparing our structures with others from
the literature we were able to come up with an explanation for the
ability of AMP-PNP to catalyse one round of the reaction cycle. In
this explanation the enzyme protonates the imino (P-N = P) form
of AMP-PNP to give the more common imido form (P-NH-P).
This insight allowed a novel mechanism for ATP hydrolysis to be
proposed. In this fully dissociative mechanism for ATP hydrolysis
a main-chain NH moves to protonate ATP, causing it to dissociate
into ADP and Pi . To the best of our knowledge this is the first
time a main-chain amide has been proposed to play a direct role
in catalysis. Moreover, this mechanochemical mechanism may
have a broad applicability to proteins such as F1 -ATPase and Gproteins such as Ras, as well as proteins involved more directly
in movement. As well as providing insights into the coupling of
ATP hydrolysis and domain movement in M. tuberculosis ATPase
gyrase, these studies open up new possibilities for the rational
design of covalent inhibitors against DNA gyrase, HSP90 and
possibly other challenging, but important, drug targets.
AUTHOR CONTRIBUTION
In GlaxoSmithKline, Alka Agrawal carried out cloning, protein expression, purification,
crystallization experiments, refinement and activity assays. Claus Spitzfaden performed
AUC and ASEC. Benjamin Bax solved structures, completed refinements and devised
dissociative mechanism. In Paris, Claudine Mayer conceived and supervised the work.
Mélanie Roué carried out cloning, protein expression, purification, activity assays,
crystallization experiments, AUC experiments, crystallographic data collection, and
refinement with the help of Claudine Mayer. Alka Agrawal, Mélanie Roué, Benjamin Bax
and Claudine Mayer wrote the paper with assistance from Stéphanie Petrella, Alexandra
Aubry and Michael Hann.
FUNDING
The work carried out in Paris was supported by the Pasteur Institute [Programmes
Transversaux de Recherche number 367]. A. Agrawal was funded by a National Science
Foundation International Research Fellowship [grant number INT-0202606].
ACKNOWLEDGEMENTS
For experimental assistance we thank members of the Crystallisation and Crystallography,
and the Biophysics of Macromolecules and their Interactions Platforms at the Pasteur
Institute and members of Biological Reagents and Assay Development department within
GlaxoSmithKline. We also thank Hélène Munier-Lehman for help with the ATPase assays,
c The Authors Journal compilation c 2013 Biochemical Society
272
A. Agrawal and others
Robert Nolte and Nigel Moriarty for computational help, and Oliver Smart, Jérémie Piton
and Olivier Poch for helpful discussions.
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Received 15 April 2013/4 September 2013; accepted 9 September 2013
Published as BJ Immediate Publication 9 September 2013, doi:10.1042/BJ20130538
c The Authors Journal compilation c 2013 Biochemical Society
Biochem. J. (2013) 456, 263–273 (Printed in Great Britain)
doi:10.1042/BJ20130538
SUPPLEMENTARY ONLINE DATA
Mycobacterium tuberculosis DNA gyrase ATPase domain structures suggest
a dissociative mechanism that explains how ATP hydrolysis is coupled to
domain motion
Alka AGRAWAL*1 , Mélanie ROUɆ‡§1 , Claus SPITZFADEN*, Stéphanie PETRELLA†‡§, Alexandra AUBRY¶**, Michael HANN*,
Benjamin BAX*2 and Claudine MAYER†‡§
*Platform Technology Sciences, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K., †Unité de Microbiologie Structurale,
Institut Pasteur, 75015 Paris, France, ‡URA 2185, CNRS, 75015 Paris, France, §Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, 75015 Paris, France, UPMC Université
Paris 06, ER5, EA 1541, Laboratoire de Bactériologie-Hygiène, Paris, France, ¶AP-HP, Hôpital Pitié-Salpêtrière, Laboratoire de Bactériologie-Hygiène, Paris, France, and **Centre
National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Paris, France
Figure S1
Comparison of two possible mechanisms for ATP hydrolysis by M. tuberculosis DNA gyrase
(A) A mechanism in which the ATP hydrolysis is initiated when a water (or OH − ion) makes a nucleophilic attack on the phosphate (associative). This scheme is essentially that proposed in [1], an
excellent paper dissecting the ATP turnover of a GHL ATPase. (B) A mechanism in which the hydrolysis is initiated when the oxygen between the β- and γ -phosphates is protonated. This causes
ATP to dissociate into ADP and a metaphosphate ion [PO3 ] − .
1
These authors contributed equally to this work.
To whom correspondence should be addressed (email [email protected]).
Co-ordinates and structure factor files for the M. tuberculosis GyrB ATPase domain have been deposited in the PDB under the accession codes 3ZKB,
3ZKD and 3ZM7
2
c The Authors Journal compilation c 2013 Biochemical Society
A. Agrawal and others
Figure S2
Common protonation states for Pi , ADP and ATP with pK values, and protonation and tautomeric states of AMP-PCP and AMP-PNP
A is adenosine. Adenosine is uncharged between pH 4.8 and 12. The common protonation states of Pi , ADP and ATP are shown [3,4]. In the presence of bound Mg2 + ions the more negatively
charged forms can be stabilized [5]. Solution NMR studies suggest that AMP-PNP probably has the hydrogen on the bridging nitrogen [6]. Although E. coli alkaline phosphatase can remove the
terminal phosphate of AMP-PNP [7], most enzymes do not efficiently hydrolyse AMP-PNP.
c The Authors Journal compilation c 2013 Biochemical Society
ATP hydrolysis by the M. tuberculosis DNA gyrase ATPase domain
Figure S3 The 1.87 Å human Topo IIA structure with AMP-PNP is consistent
with a bridging imino (P-N = P), but not a bridging imido (P-NH-P)
(A) The 1.87 Å human Topo IIA structure with AMP-PNP (PDB code 1ZXM). Distances from
main-chain nitrogens of Gly164 and Arg162 are indicated by broken lines (estimated error ∼0.1 Å
based on two molecules in asymmetic unit). (B) The original AMP-PNP was removed, hydrogens
were added to the protein and a difference map was calculated (insert 5 σ F o − F c ). The imino
P-N = P form of AMP-PNP was modelled [8] into the F o − F c map using PHENIX [9] and
displayed in Coot [10]. Distances from hydrogens on main-chain nitrogens of Gly164 and Arg162
are indicated by broken lines. (The hydrogen on the main-chain nitrogen of Asn163 is some
2.9 Å from the bridging nitrogen). (C) Superimposition of (A) and (B).
Figure S4
ATP activity assays on M. tuberculosis DNA gyrase
(A) ATPase activity of the M. tuberculosis DNA gyrase as a function of protein concentration.
Rates are initial velocities. The substrate (ATP) concentration was 1 mM, and the subunits GyrA
and GyrB were mixed in equimolar concentrations. (B) ATPase activity of the M. tuberculosis
DNA gyrase in the presence and absence of DNA (relaxed pBR322 at 5 μg/ml). Rates are
initial velocities. The substrate (ATP) concentration was 1 mM, and the subunits GyrA and GyrB
were mixed in equimolar concentrations. Increased ATPase activity in the presence of DNA has
previously been shown for E. coli DNA gyrase, yeast, human and protozoan topoisomerases
II, and the P. falciparum DNA gyrase [11–17]. (C) ATPase activity of the M. tuberculosis DNA
gyrase as a function of substrate (ATP) concentration at a constant enzyme concentration. Rates
are initial velocities. The subunits GyrA and GyrB both at 5 μM. The K m was calculated by the
Lineweaver–Burk plot. Initial rates were correlated with a phosphate calibration curve.
c The Authors Journal compilation c 2013 Biochemical Society
A. Agrawal and others
Figure S5
F o − F c ( + 2.5 σ ) maps showing difference density for the six AMP-PCPs in the asymmetric unit
The final structure is shown with a map phased on the original molecular replacement solution from which the ATP lid, N-arm and, in molB, residues from the transducer domain have been deleted.
The map shows the density for all AMP-PCPs, but no density for the ATP lid. For molA the AMP-PNP structure is shown superimposed.
c The Authors Journal compilation c 2013 Biochemical Society
ATP hydrolysis by the M. tuberculosis DNA gyrase ATPase domain
Figure S6
Comparison of structures of type IIA topoisomerases with AMP-PNP
Three orthogonal views of (A) M.tuberculosis GyrB (the present study), (B) 2.3 Å E.coli GyrB (PDB code 1EI1) and (C) 2.1 Å structure E. coli Topo IV (PDB code 1S16). (D) Superimposition of
(A–C) (the structures are superimposed using one GHKL domain, indicated by an arrow in the centre of A). (E) The 1.87 Å human Topo IIA (PDB code 1ZXM). Because the superimpositions in this
Figure were done on a single GHKL domain (indicated by an arrow in A), this Figure emphasizes differences between the structures.
c The Authors Journal compilation c 2013 Biochemical Society
A. Agrawal and others
Figure S7
Structure-corrected sequence alignment of the ATPase domain from type IIA topoisomerases
The sequence names are as follows: M.tuberculosis_GyrB , M. tuberculosis DNA gyrase; M.leprae_GyrB , M. leprae DNA gyrase; 1EI1_E.coli_Gyr , E. coli DNA gyrase (PDB code 1EI1);
1S16_E.coli_TopoIV , E. coli Topo IV (PDB code 1S16); 1PVG_S.cerev_TopoII , S. cerevisiae Topo II (PDB code 1PVG); and 1ZXM_Human_TopoII , human Topo II (PDB code 1ZXM). α-Helices and
β-strands are shown above the sequences as red and blue cylinders respectively, and are defined from the E. coli DNA gyrase ATPase domain (PDB code 1EI1) from PDBSum. The ATP lid is indicated
by a green arrow. The vertical green bar delimits the end of the GHKL and the beginning of the transducer sub-domains. The insert regions (32 or 34 residues) of M. tuberculosis and M. leprae are
indicated in brown. The two constructs used in the present paper both had the M. tuberculosis GyrB sequence shown in alignment, but they had different N-terminal tags: Mtb GyrB47C1 had a 19
residue tag MGHHHHHHLEVLFQ/GPLGS and Mtb GyrB47C2 had a 15 residue N-terminal tag with MAHHHHHHVDDDDK/ with the cleavage site just prior to Val1 (where / represents a cleavage site).
c The Authors Journal compilation c 2013 Biochemical Society
ATP hydrolysis by the M. tuberculosis DNA gyrase ATPase domain
Figure S8
The insert region of the M. tuberculosis ATPase domain is involved in crystal contacts
(A) View of the Mtb GyrB47–AMP-PNP dimer showing positions of the largely disordered insert regions (framed in red). (B) Superimposition of the GHKL domain of P. falciparum HSP90 (PDB
code 3PEH) showing an insert at the same position. (C) Dimer–dimer interactions observed in all Mtb GyrB47–AMP-PNP dimers [shown between the AB and EF chains in the P 1(8) form]. Residues
from the insert are in brown. (D) The insert region is also close to crystal contacts in the Mtb GyrB47–AMP-PCP structure. The first residue of β10 and last residue of β11 are represented in brown
CPK (Corey–Pauling–Koltun).
Figure S9
AUC of the M. tuberculosis GyrB ATPase domain
AUC traces are shown of untagged Mtb GyrB47C2 at 17 μM (bottom) and Mtb GyrBC2 in the
presence of 2 mM MgCl2 and 2 mM AMP-PCP (top).
c The Authors Journal compilation c 2013 Biochemical Society
A. Agrawal and others
Figure S10 Dissociative reaction schemes for the (A) synthesis of ATP by the GyrB ATPase domain and (B) for the transfer of a phosphate from a histidine
to an aspartic acid residue in two-component signal transduction
Figure S11
Comparison of transition state complexes of Ras and F1 -ATPase with the AMP-PNP structure of M . tuberculosis GyrB47
(A) Ras (cyan carbons)/RasGAP(dark green carbons) complexed with ADP and AlF3 (PDB code 1WQ1). The Mg2 + is shown as a small green sphere. Arg789 from RasGAP accelerates ATP hydrolysis.
(B) F1 -ATPase with ADP and BeF3 . The Mg2 + is shown as a small green sphere. Two F1 -ATPase subunits are indicated by carbons in different shades of green. (C) Mtb GyrB47–AMP-PNP. (D)
Superimposition of (A–C).
c The Authors Journal compilation c 2013 Biochemical Society
ATP hydrolysis by the M. tuberculosis DNA gyrase ATPase domain
Figure S12
A simplified dissociative mechanism for the hydrolysis of GTP by Ras
(A) In the presence of wild-type Ras, Gln61 helps to position a water, so that it can attack the metaphosphate ion [PO3 ] − , once it is formed. Movement of the NH of Gly13 past the bridging oxygen is
responsible for the initial protonation, and by the time the glycine has moved again (to a position where it can take back the proton from GDP) the phosphate ion has formed. (B) In the presence of
an Ala61 mutant the water is not optimally positioned, so the metaphosphate ion is likely to be re-attacked by GDP before the water can attack.
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