Seventh International Meeting on AAA Proteins
Dissecting the ATP hydrolysis pathway of bacterial
enhancer-binding proteins
Daniel Bose*, Nicolas Joly†, Tillmann Pape*, Mathieu Rappas*, Jorg Schumacher†, Martin Buck† and Xiaodong Zhang*1
*Division of Molecular Bioscience, Faculty of Natural Sciences, Imperial College London, South Kensington, London, SW7 2AZ, U.K., and †Division of Biology,
Faculty of Natural Sciences, Imperial College London, South Kensington, London, SW7 2AZ, U.K.
Abstract
bEBPs (bacterial enhancer-binding proteins) are AAA+ (ATPase associated with various cellular activities)
transcription activators that activate gene transcription through a specific bacterial σ factor, σ 54 . σ 54 –RNAP
(RNA polymerase) binds to promoter DNA sites and forms a stable closed complex, unable to proceed to
transcription. The closed complex must be remodelled using energy from ATP hydrolysis provided by bEBPs
to melt DNA and initiate transcription. Recently, large amounts of structural and biochemical data have
produced insights into how ATP hydrolysis within the active site of bEBPs is coupled to the re-modelling
of the closed complex. In the present article, we review some of the key nucleotides, mutations and
techniques used and how they have contributed towards our understanding of the function of bEBPs.
Introduction
bEBPs (bacterial enhancer-binding proteins) are AAA+
(ATPase associated with various cellular activities) family
members involved in regulating bacterial gene expression.
Regulated transcription in bacteria requires multisubunit
RNAP (RNA polymerase) to interact with σ factors, a
family of proteins that bind to RNAP forming holoenzymes
and direct it to promoter DNA [1,2]. bEBPs are essential for
transcription initiation using the major variant σ factor, σ 54 ,
commonly involved in tightly regulated bacterial responses
to stress [3–5]. This is functionally analogous to enhancerdependent initiation of eukaryotic RNA polymerase II,
which requires an input of energy from ATP hydrolysis
provided by TFIIH (transcription factor IIH) [6,7]. ATP
hydrolysis by bEBPs provides energy for remodelling
the σ 54 –RNAP closed complex, resulting in further DNA
melting and loading of the template strand of DNA into the
RNAP active site [8,9]. Energy is transferred to the closed
complex through a physical interaction between σ 54 and the
AAA+ domain of the bEBP [10,11].
A number of bEBPs are well characterized, including
NtrC, ZraR, PspF, NorR, DctD, NifA and HrpR/S [12,13].
They typically comprise three domains. An N-terminal
regulatory (R) domain acts either positively or negatively on
ring formation and ATPase activity [14]. Positive regulation
(e.g. NtrC and ZraR) is when the modification of the R
domain actively promotes oligomerization, whereas, in
negative regulation, the modification of R domain releases
Key words: ATPase associated with various cellular activities (AAA+), bacterial enhancer-binding
protein, nucleotide analogue, σ 54 , transcriptional regulation.
Abbreviations used: AAA, ATPase associated with various cellular activities; ATP[S], adenosine
5 -[γ -thio]triphosphate; bEBP, bacterial enhancer-binding protein; EM, electron microscopy;
p[NH]ppA, adenosine 5 -[β,γ -imido]triphosphate; RNAP, RNA polymerase; SAXS, small-angle
X-ray scattering; SRH, second region of homology; WAXS, wide-angle X-ray scattering.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2008) 36, 83–88; doi:10.1042/BST0360083
the inhibition of oligomerization (e.g. NtrC1 and DctD)
[15–18]. In some cases, the R domain is dispensed with,
and regulation is achieved through an interaction with a
secondary protein (e.g. PspF with PspA and HrpR/S with
HrpV) [19–21]. The C-terminal DNA-binding domain
enables specific promoter recognition by allowing bEBPs to
bind to enhancer-like sequences located approx. 100–150 bp
upstream of the transcription start site [3,22]. bEBPs bound
to upstream DNA contact the closed σ 54 –RNAP complex by
a DNA-looping mechanism, often aided by DNA-bending
proteins [23]. Interactions with DNA may also promote
oligomerization of the AAA+ domain (e.g. NorR) [24].
The AAA+ domain in isolation is often sufficient to
activate σ 54 -dependent transcription in vitro and in vivo
[25,26]. Apart from the well-conserved AAA+-specific
motifs Walker A (GXXXXGKT/S), Walker B (DE,
where is hydrophobic) and the SRH (second region of
homology) motif, bEBPs also contain the highly conserved
signature GAFTGA motif, which mediates interactions with
σ 54 [27–31]. Nucleotide binding occurs at the interface of
two subunits, and residues from adjacent subunits contribute
to hydrolysis, partly explaining the dependence of ATP
hydrolysis activity on oligomerization [5,32,33]. The AAA+
domain also contains the L1 and L2 loops, which are specific
to bEBPs and undergo a series of movements in response to
stages of the ATP hydrolysis cycle [10,34]. L1 contains the
GAFTGA motif at its tip and forms an insertion in the third
helix of the AAA+ domain, present as an uninterrupted helix
in other AAA+ family members [27]. L2 is present at the Cterminus of the fourth helix, and is thought to be responsible
for co-ordinating the movements of the L1 loop [10]. bEBPs
are members of a subclass of AAA proteins known as the
pre-sensor I β-hairpin superclade, which share the insertion
of a β-hairpin in the position of loop L2, and include
the helicases RuvB, Ltag and MCM (mini-chromosome
maintenance), as well as proteases HslU, ClpX and Lon [35].
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The nucleotide state in the active site is communicated
to the L1 and L2 loops, causing loop movements and affecting
interactions between the bEBP and σ 54 –RNAP [36,37]. The
transition from closed to open complex is likely to involve at
least two transient intermediate states, possibly as a result of
changing interactions with the GAFTGA motif during the
ATP hydrolysis cycle [38–40]. A wide variety of techniques
and approaches have been employed to study the intricate
events that accompany the ATP hydrolysis cycle within
bEBPs. In this review, we describe our current understanding
of the bEBP-mediated transition from closed to open
complex, and examine some of the important techniques,
nucleotide analogues and mutations employed to dissect the
stages of the ATP hydrolysis cycle that are responsible for
this transition.
Structural studies
A number of high-resolution structures of bEBPs have
revealed large amounts of information regarding the coupling
of nucleotide hydrolysis to functional interactions with σ 54
within bEBPs, and, together with information from other
AAA+ family members, have enabled the design of rational
mutations to study these processes further. To date, structures
exist for NtrC1, PspF and ZraR in different nucleotide states
and varying mutant forms [10,16,27,34]. A number of the
nucleotide-bound structures of PspF were obtained by soaking crystals with nucleotides, detailing the localized changes
occurring in the active site during hydrolysis, and suggesting a
network of interactions linking the active site to loops L1 and
L2 [10,36]. Lower-resolution methods such as SAXS (smallangle X-ray scattering)/WAXS (wide-angle X-ray scattering)
and single-particle cryo- and negative-stain EM (electron
microscopy) have been used in conjunction with nucleotide
analogues to provide a glimpse of the macromolecular
changes occurring during the hydrolysis cycle, and the cryoEM structure of PspF in complex with σ 54 highlighted
the importance of the L1/L2 loops for coupling nucleotide
hydrolysis to remodelling of the closed complex [10,15,41].
Nucleotide states
Ground state (ATP/ADP–BeF/p[NH]ppA/ATP[S])
Binding of ATP in the active site is communicated through a
network of interactions causing a relocation of Linker 1 and
subsequent rotation of the third helix of the AAA+ domain
[36]. This causes the L1/L2 loops to move upwards, positioning the GAFTGA motif in an exposed conformation able to
interact with σ 54 (Figure 1A) [10]. X-ray structures of PspF
bound to ATP were obtained by soaking crystals of wild-type
PspF with ATP in the absence of Mg2+ to prevent hydrolysis,
or by using the hydrolysis-deficient mutant form of PspF
(PspFR227A ) in the presence of Mg2+ . The structure of wildtype PspF soaked with the ground-state analogue p[NH]ppA
was also solved [9]. The structures show the localized interactions made between ATP and the active site of PspF, notably
the Walker B aspartate residue (Asp107 ) positions a water
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the glutamate residue (Glu108 ) senses the γ -phosphate of the
bound nucleotide and forms a tight interaction with
the highly conserved asparagine residue (Asn64 ) at the Nterminus of Linker 1 (Figure 1B) [36]. Although crystal
lattice constraints limit the observation of larger-scale
conformational changes, studies using NtrC1 with ADP–BeF
as a ground-state analogue indicate that the initial contact
with σ 54 is indeed formed during the ground state [41].
ADP–BeF causes a pronounced raising of the L1/L2 loops in
NtrC1 that can be observed using SAXS/WAXS analysis, a
result consistent with the local changes observed in the ATPbound crystal structure of PspF [36,41]. Furthermore, σ 54
can form complexes with NtrC1 and PspF in the presence
of ADP–BeF [41]. Interestingly, p[NH]ppA (adenosine
5 -[β,γ -imido]triphosphate) and ATP[S] (adenosine 5 -[γ thio]triphosphate) do not cause any loop movements
observable by SAXS/WAXS, although p[NH]ppA can
stabilize complexes between σ 54 and NtrC1 or PspF [41]
(Table 1). The results suggest that ATP binding can cause
the release of the L1/L2 loops and enables an initial unstable
interaction with σ 54 .
Transition state (ADP–AlFx )
Use of the ATP hydrolysis transition-state analogue ADP–
AlFx (where x is either 3 or 4) has been critical for understanding the function of bEBPs (Table 1). The ability of ADP–
AlFx to form stable complexes with σ 54 led to observations
implicating the importance of a direct interaction between
GAFTGA and σ 54 -region I for the isomerization of the
closed complex [8,32,42]. Most importantly, the stabilizing
effect of ADP–AlFx on the complex between PspF and σ 54
enabled reconstruction of this complex using single-particle
cryo-EM [10]. Together with the X-ray structure of PspF, the
reconstruction revealed that at the point of ATP hydrolysis,
the L1/L2 loops elevate above the plane of the hexameric ring
and make significant interactions with σ 54 , probably through
the GAFTGA motif [10,11]. Reconstructions of NtrC and
NtrC1 bound to ADP–AlFx , solved using SAXS/WAXS
and negative-stain EM, show a pronounced ring of density
above the plane of the oligomeric bEBP, corresponding to
the raised L1/L2 loops in the transition state [15,41]. Stable
complexes with σ 54 have been observed in the presence
of ADP–AlFx for NtrC1 and NifA, and stable complexes
form between PspF and σ 54 –RNAP [41,42]. Interestingly, the
NtrC1–σ 54 complex formed with ADP–AlFx is significantly
more stable than that formed with ADP–BeF, suggesting that
the initial complex formed with the ground state is stabilized
upon transition-state formation [40,41]. ADP–AlFx -bound
PspF has a greatly reduced ATP-hydrolysis activity and
is incapable of initiating transcription [42]. However, it is
capable of inducing conformational changes of both σ 54 –
RNAP and promoter DNA within the closed complex
similar to those observed during open complex formation
[39,40]. Consequently, the interaction stabilized by ADP–
AlFx is likely to represent a genuine intermediate on the
isomerization pathway between closed and open complexes.
Seventh International Meeting on AAA Proteins
Figure 1 Conformational changes during ATP hydrolysis
Structures of PspF1−275 bound to ATP (A, C) PDB code 2C96/2C9C and ADP (B, D) PDB code 2C99/2C9C. Important motifs are
highlighted: Walker A (red), Walker B (blue), Sensor I (orange), R-finger (green), Sensor II (magenta), Linker 1 (pale green),
Loops L1 (red) and L2 (blue), conformational signalling between Asn64 and L1 (yellow). Monomeric PspF1−275 in (A) ATP
state and (B) ADP state, showing raised L1 conformation in ATP state and lowered conformation in ADP state. Active-site
interactions with (C) ATP and (D) ADP. (E) Switching mechanism of Walker B Glu108 . In ATP state, Glu108 (blue) interacts with
Asn64 . In ADP state Glu108 (teal) interacts with Thr148 .
Table 1 Nucleotide states
Nucleotides and analogues, the state modelled and effect when bound to bEBPs [36,41,43]
Nucleotide
Nucleotide state
Properties when bound to bEBP
ATP
ADP–BeF
Ground state (ATP)
Ground state (ATP)
Promotes hexamerization; high concentrations inhibit catalytic activity
L1/L2 loops in raised position to contact σ 54 . Stabilizes complex with σ 54 in NtrC1 and PspF
p[NH]ppA
ATP[S]
Ground state (ATP)
Slow hydroysis
Can stabilize complex of NtrC1 and σ 54
No complex stabilization
ADP–AlFx
Hydrolysis transition state
L1/L2 loops in raised position. Forms stable complex with σ 54 with improved stability over ADP–BeF
complex. Reduced hydrolysis activity. Can re-model closed complex, forming the first of two
proposed intermediate complexes
ADP
Post-hydrolysis (ADP)
L1/L2 loops in lowered conformation. High concentrations inhibit hydrolysis, but maximum catalytic
activity when bound at physiological concentrations with ATP in PspF
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Post-hydrolysis (ADP)
The ADP-bound state has been a more tractable prospect to
study structurally, with crystal structures solved for PspF,
NtrC1 and ZraR [10,16,34,36]. The structure of ADP-bound
PspF indicates that release of the γ -phosphate causes a
90◦ rotation of the Glu108 side chain, which forms a new
interaction with the Sensor I threonine residue (Thr148 )
(Figure 1E) [36]. This is communicated through Asn64 and
Linker 1, causing a rotation of the third helix [36]. The
structure of ADP-bound NtrC1 shows the L1/L2 loops
locked into a position pointing towards the central pore of the
bEBP, unavailable for σ 54 interactions (Figure 1B) [34]. This
conformation is maintained by hydrophobic interactions
between the phenylalanine residue of GAFTGA and
hydrophobic residues in L2 and in the third helix [34]. The
Walker B glutamate residue appears to act as an atomic switch
causing movement of the L1/L2 loops from a raised position
in the ATP and ADP–AlFx states to the lowered position observed in the ADP state [27,36]. Binding of ADP can
stimulate formation of PspF hexamers and has a stimulatory
effect on the ATP-hydrolysis activity of PspF [43]. This
partly reflects the role of ADP in hexamer formation, but
also involves intersubunit communication [43].
Functional determinants
Walker B motif
Mutations of the Walker B motif (Asp-Glu) have dramatic
effects on the ATP hydrolysis activity of AAA proteins [27].
The aspartate residue is highly conserved and principally
involved in hydrolysis, consequently mutations of the residue
typically result in hydrolysis-deficient proteins [33,44]. In the
absence of nucleotide, the aspartate residue appears to negatively regulate hexamer formation, as substitutions of Asp107
in PspF can result in formation of constitutive hexamers
[40]. Mutations of the glutamate residue can uncouple
nucleotide hydrolysis from functional output, preventing
hydrolysis, but not the conformational movements resulting
from nucleotide binding [45,46]. The mutation of PspF Glu108
to alanine or glutamine (E108A or E108Q) results in mutant
forms capable of forming nucleotide-independent hexamers,
albeit with a greatly reduced nucleotide hydrolysis activity
[40]. More significantly, the Glu108 mutants are also capable of
forming a relatively stable complex with σ 54 and σ 54 –RNAP
in the presence of ATP, a state only obtainable with ADP–BeF
and ADP–AlFx in wild-type protein [40–42]. Observation of
a stable complex with σ 54 when ATP is bound, but not hydrolysed, is consistent with the complexes seen with NtrC1,
PspF and σ 54 when bound to ADP–BeF [40,41]. The side
chain of Glu108 in PspF switches between interactions with
Asn64 and Thr148 in response to ATP hydrolysis, controlling
the movements of L1 and L2 [36,47]. The ability of E108A to
bind stably to σ 54 suggests that the raised conformation of L1
and L2 is not wholly dependent on a tight interaction between
Glu108 and Asn64 [40]. In contrast with the ADP–AlFx complex, the Glu108 variant complexes are transcriptionally active,
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Authors Journal compilation despite having a low rate of ATP hydrolysis [40]. Similar results have been observed for mutation of the equivalent residue
in NtrC1 (Glu239 ) to alanine [41]. Mutations of Glu108 in
PspF also enabled identification, on the basis of altered DNA
conformations, of at least two of the intermediate complexes
formed en route to full open complex formation. The first
is more similar to that observed with the trapped ADP–
AlFx complex, whereas the second represents a conformation
closer to that of the open complex [37,39,40]. The presence of
a side chain at position 108 appears to be essential for formation of these isomerized complexes, reflecting the importance
of a switching mechanism during ATP hydrolysis [40].
SRH (Sensor I, R-finger)
SRH contains the sensor I motif and the R-finger, a catalytic arginine residue that acts in trans between adjacent protomers [30,31]. Mutation of the R-finger (PspF Arg168 ) to
alanine abolishes nucleotide hydrolysis, but enables PspF
to form nucleotide-independent hexamers [33]. Mutation
of either Sensor I Thr148 or Asn149 in PspF to alanine also
results in a reduced ATP-hydrolysis activity [47]. However,
the T148A mutation is additionally defective in making
stable interactions with σ 54 and consequently for open
complex formation, highlighting the potential importance
of the interaction between Sensor I (Thr148 ) and the Walker
B residue Glu108 for communicating nucleotide state to
the L1/L2 loops [36,47]. Interestingly, this interaction
(which occurs through a water molecule present in every
nucleotide-bound structure) could provide one structural
basis for ATP-hydrolysis-driven movements of the L2 loop.
T148A mutants display similar functional phenotypes to
those observed with L2 loop mutants R131A and V132A,
which do not interact with σ 54 (Table 2). Movements of L2
in response to the post-hydrolysis ADP state initiated by a
Glu108 –Thr148 interaction and propagated through a second
signalling pathway could amplify the effect of the ‘power
stroke’ as L1 and L2 relocate to the lowered position [47].
Order of hydrolysis
The cryo-EM structure of the PspF–ADP–AlFx –σ 54 complex
reveals an asymmetric distribution of contacts made to σ 54 ,
raising the question of how hexameric bEBPs interact with
monomeric asymmetric σ 54 –RNAP [10]. This asymmetry
immediately disfavours a concerted mechanism, where
every subunit has the same nucleotide state, in favour of
either a stochastic, sequential or rotational hydrolysis mechanism, characterized by heterogeneous nucleotide occupancy.
Recent work with PspF has addressed how the order of
hydrolysis and interactions with substrate affect hydrolysis. ADP has a stimulatory effect on the ATP-hydrolysis
activity of PspF, which, together with the observation that
saturating concentrations of ATP inhibit hydrolysis, suggests
heterogeneous nucleotide occupancy and communication
of nucleotide states between subunits [43]. Indeed,
heterogeneous nucleotide occupancy in PspF correlates with
maximal catalytic activity at physiological concentrations of
Seventh International Meeting on AAA Proteins
Table 2 Functional determinants
Key mutations of bEBP motifs, mutant phenotypes and potential as tools for studying function [11,33,36,40,47].
Residue (PspF)
Mutations
Mutant phenotype
Potential as tool
Walker A
Lys42
K42A
Deficient for ATP binding, hydrolysis, hexamer formation
–
and initiation
Walker B
Asp107
Glu108
D107A, D107E
Decreased ATP hydrolysis. D107E shows increased ATP
Nucleotide-independent hexamers for
E108A, E108D, E108Q
binding. Forms constitutive hexamers
Increased ATP binding. Decreased hydrolysis and decreased
rate of initiation. E108A and E108Q form concentration-
structural studies
A/Q complexes with σ 54 possibly suitable
for structural studies. Q has very low
independent hexamers and stable complexes with σ 54 in
ATP state. E108D and E108Q can isomerize DNA in closed
complex, forming at least two intermediate complexes.
ATPase activity, therefore best
candidate for studies of ATP state
E108D has improved efficiency of ATP use compared with
wild-type
R-finger
Arg168
R168A
Reduced ATP binding, defective for hydrolysis and initiation. Nucleotide-independent hexamers for
Forms nucleotide-independent hexamers
structural studies
T148A
Slightly reduced hydrolysis, defective for transcription
activation. Deficient for isomerization of the closed
Studies of possible second conformational
signalling mechanism
complex, but able to bind σ 54
Slightly reduced hydrolysis, able to bind σ 54 and isomerize
closed complex
–
Slightly reduced hydrolysis, defective for transcription
activation. Deficient for isomerization of the closed
Studies of possible second conformational
signalling mechanism
complex, but able to bind σ 54
Slightly reduced hydrolysis, defective for transcription
activation. Deficient for isomerization of the closed
Studies of possible second conformational
signalling mechanism
Sensor I
Thr148
Asn149
Loop 2
Arg131
Val132
N149A, N149S
R131A
V132A
complex, but able to bind σ 54
GAFTGA
Thr86
T86S
Deficient for initiation with wild type σ 54 , but rescued by
Studies of bEBP–σ 54 interactions
mutants of region I
Sensor II
Arg227
R227A
Structural studies with ATP and Mg2+
Binds ATP, but deficient for hydrolysis, nucleotideindependent hexamer formation and initiation
nucleotide [43]. This argues against concerted mechanisms
and also stochastic mechanisms, as nucleotide state is
communicated between adjacent subunits [43]. Rotational
or sequential mechanisms are therefore most likely for
bEBPs. A non-symmetric mechanism in bEBPs would
permit asymmetrical contacts to form with σ 54 in the closed
complex, while reducing cycles of unproductive hydrolysis.
Summary
In summary, structural and biochemical studies of bEBPs
have revealed a multistep process during the ATP hydrolysis
cycle, which correlates with intermediate complexes
formed during open complex formation. Conformational
signalling propagating from the Walker B glutamate residue
communicates changes in nucleotide state to the L1/L2 loops.
Hexameric bEBPs probably use a non-concerted mechanism
for ATP hydrolysis. Studies of the detailed conformational
changes and co-ordination within the hexameric ring during
an ATP hydrolysis cycle, and the interactions made with
σ 54 –RNAP holoenzyme that ultimately lead to DNA
melting and open complex formation, remain the great
challenges for the coming years.
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Received 29 October 2007
doi:10.1042/BST0360083