Enzymology and Ecology of the Nitrogen Cycle
The role of effector molecules in signal
transduction by PII proteins
Martha Radchenko and Mike Merrick1
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, U.K.
Abstract
PII proteins are one of the most widely distributed signal transduction proteins in Nature, being ubiquitous
in bacteria, archaea and plants. They act by protein–protein interaction to control the activities of a wide
range of enzymes, transcription factors and transport proteins, the great majority of which are involved
in cellular nitrogen metabolism. The regulatory activities of PII proteins are mediated through their ability
to bind the key effector metabolites 2-OG (2-oxoglutarate), ATP and ADP. However, the molecular basis
of these regulatory effects remains unclear. Recent advances in the solution of the crystal structures of
PII proteins complexed with some of their target proteins, as well as the identification of the ATP/ADPand 2-OG-binding sites, have improved our understanding of their mode of action. In all of the complex
structures solved to date, the flexible T-loops of PII facilitate interaction with the target protein. The effector
molecules appear to play a key role in modulating the conformation of the T-loops and thereby regulating
the interactions between PII and its targets.
Introduction
The structures of PII proteins
The PII signal transduction proteins were first identified in
1969 during studies of post-translational modification of
the activity of Escherichia coli GS (glutamine synthetase)
[1]. At that time, two protein fractions, PI and PII , were
described: PI subsequently being determined to contain both
the adenylyltransferase (GlnE) responsible for adenylylation
of GS, and a uridylyltransferase (GlnD) responsible for the
reversible uridylylation of PII . PII regulated the activity of
GlnE and was later shown to be encoded by glnB. PII proteins
are now recognized as one of the most widely distributed
signal transduction proteins in Nature, being ubiquitous in
bacteria, archaea and plants [2]. Many bacteria and archaea
encode multiple PII proteins, whereas only a single copy is
present in the cyanobacteria and in plants [2].
PII proteins function by protein–protein interaction,
whereby they have been shown to control the activities of
a wide range of targets, including enzymes, transcription
factors and membrane transport proteins. These targets are
almost invariably involved in cellular nitrogen metabolism,
and hence PII proteins are considered to be pivotal regulators
of nitrogen metabolism in most prokaryotes. More recently,
it has also been proposed that their functions may extend
to the co-ordination of nitrogen and carbon metabolism
and to sensing of cellular energy status [3]. The key to the
sensory properties of the PII proteins is their ability to bind
the effector molecules 2-OG (2-oxoglutarate) and ATP or
ADP. In the present review, we discuss recent advances in
our understanding of this process.
PII proteins are homotrimers with 12–13 kDa subunits that
display a highly conserved structure. The trimer is a compact
cylindrically shaped molecule from which three long exposed
loops (the T-loops) protrude (Figure 1A). The T-loops are
significantly conserved in sequence, but are structurally very
flexible and can adopt various conformations. They are
vital for PII interactions with many of their targets and
are also often sites of reversible covalent modification [4,5]. In
addition to the T-loops, PII proteins are also characterized by
three lateral inter-subunit clefts, within which are two smaller
loops (the B- and C-loops) (Figure 1). PII proteins have been
divided into three subfamilies, encoded by the genes glnB,
glnK and nifI [4]. GlnB and GlnK are closely related and are
often encoded in the same organism. A considerable number
of X-ray structures have been solved for both GlnB and
GlnK. NifI proteins are more distantly related: they occur
in nitrogen-fixing archaea and function in regulating nitrogen
fixation [6]; however, no structures of NifI proteins have been
solved. Recently, a detailed phylogenetic analysis identified a
new group of PII proteins, designated PII-NG [2]. Very little
is known about these proteins, but their structural genes are
frequently linked to genes for heavy metal transporters and it
is deduced that they may be involved in metal homoeostasis.
We will not discuss this group further in the present paper.
Key words: N-acetyl-l-glutamate kinase, crystal structure, nitrogen metabolism, 2-oxoglutarate,
PII protein, signal transduction.
Abbreviations used: 2-OG, 2-oxoglutarate; GS, glutamine synthetase; NAG, N-acetylglutamate;
NAGK, N-acetyl-l-glutamate kinase.
To whom correspondence should be addressed (email [email protected]).
1
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The interactions of effectors with PII
proteins
Early studies with Escherichia coli GlnB implicated ATP, 2OG and Mg2+ as potential effector molecules that facilitate
PII -mediated regulation. It is now apparent, from studies in
a number of systems, that ATP and 2-OG bind to PII at
micromolar concentrations and their binding is synergistic
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Figure 1 The structure of the E. coli PII protein GlnK
(A) Side view showing one possible conformation of the T-loops and the position of Tyr51 , which is the site of uridylylation in
many PII proteins. (B) View from below showing the binding site for ATP/ADP located in the inter-subunit cleft. The Figures
were drawn using the software package PyMOL (DeLano Scientific; http://www.pymol.org) with the PDB codes 1GNK (A)
and 2GNK (B).
[5,7,8]. However, as ATP binds avidly in the presence
of 2-OG, it was considered unlikely that the nucleotide
plays a regulatory role in vivo. It was only recognized
relatively recently that PII proteins could also bind ADP
and that this might be physiologically significant [3,9,10].
Biochemical studies on E. coli GlnB and GlnK showed that
ADP acted antagonistically to 2-OG [3,11]. The ability of PII
proteins to bind ATP or ADP alternately substantiated earlier
suggestions from studies in Rhodospirillum rubrum that, in
addition to sensing 2-OG levels, PII proteins might have the
capacity to sense adenylate energy charge [3,9].
The site of ATP binding was determined from the crystal
structure of E. coli GlnK co-crystallized with ATP [12]. The
nucleotide binds in the lateral clefts between the subunits,
where the B-loop from one subunit and C-loop from another
contribute to a nucleotide-binding pocket (Figure 1B). The Bloop includes a consensus sequence (G84 XXGXGK) identical
with that found in other nucleotide-binding proteins and
referred to as the Walker A motif. Key contacts to the
phosphate groups include Gly87 and Gly89 in the B-loop
and Arg101 and Arg103 from the C-loop, all of which are
highly conserved residues in the PII family. When ADP
was identified in the structure of the complex between E.
coli GlnK and the ammonia channel AmtB (see below), it
occupied the same site as ATP (Figure 2A) and made very
similar interactions, the β-phosphate of ADP occupying a
very similar position to that of the γ -phosphate in the GlnK–
ATP structure. ADP has also been identified in structures
of PII proteins from Methanococcus janaschii (PDB code
2J9D) and Thermotoga maritima (PDB code 1O51), but the
physiological significance of these structures is unclear.
The 2-OG-binding site has proved much more elusive.
In a structure of M. janaschii GlnK1, 2-OG was found
to be bound to one of the T-loops of the trimer [13]. A
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homologue) shows a more convincing location for 2-OG,
bound near to Mg-ATP within the lateral cleft (Figure 2C)
[14]. This structure fully rationalizes the synergistic binding
of 2-OG and ATP and is consistent with data from E. coli
GlnB showing that 2-OG binds even when the T-loops
are deleted [15]. The GlnZ structure now offers a basis on
which to understand the role of effector binding, namely
in influencing the conformation of the T-loops. Two key
residues in this structure are the highly conserved Gln39 and
Lys58 , each of which lie at the base of the T-loop (Figure 2C).
Both ATP and 2-OG participate in the co-ordination of the
Mg2+ ion, three bonds provided by the phosphates of ATP,
two by the 2-oxo acid moiety of 2-OG and the final one by
the Gln39 side chain. 2-OG also forms a salt bridge with Lys58
(Figure 2C). The significance of these interactions becomes
apparent from recent structures of PII proteins complexed
with some of their targets (see below). However, it should
also be noted that, whereas the roles of 2-OG and ATP/ADP
as PII effectors has been considered to be a universal feature
of these proteins, Bacillus subtilis GlnK has been reported
to bind 2-OG only weakly [16] and Archaeoglobus fulgidus
GlnK2 shows no 2-OG binding [17].
PII protein targets
The initial studies of PII were in the context of regulation of
GS activity and specifically the regulation of adenylyltransferase activity by GlnB. Since then PII proteins have been
found to regulate a wide variety of targets including enzymes,
transcription factors and membrane transport proteins (see
[4,6,18] for reviews). Bioinformatic analysis of the likely
evolution of PII proteins supports the concept that the
ancestral PII evolved in association with the ammonia channel
Enzymology and Ecology of the Nitrogen Cycle
Figure 2 Binding of effectors to PII proteins and the influence of 2-OG
(A) ADP bound to E. coli GlnK when complexed with AmtB; (B) S. elongatus PII with bound Mg-ATP; and (C) 2-OG and Mg-ATP
bound to A. brasilense GlnZ. Note how the T-loop residue Gln39 or Glu44 can form a salt bridge with Lys58 in the absence
of 2-OG (A and B). This interaction is disrupted by the binding of 2-OG (C) with a consequent effect on the conformation of
the T-loop. The Figures were drawn using the software package PyMOL (DeLano Scientific; http://www.pymol.org) with the
PDB codes 2NUU (A), 2V5H (B) and 3MHY (C).
AmtB [2,19]. The key role of GlnK proteins in forming a
complex with the trimeric AmtB to regulate ammonia influx
into cells rationalizes the trimeric nature of PII proteins.
However, it is clear that the regulatory properties of PII
proteins have subsequently been adopted to co-ordinate
regulation of a whole range of cellular activities [4,18].
This means that PII proteins are able to interact with a
wide variety of different protein structures and that the
mechanisms of these interactions are likely to be equally
varied. Of particular interest in this regard is the fact that
many of the PII targets are not trimeric, raising questions
about the likely stoichiometry of many of these interactions.
A detailed understanding of the molecular mechanisms
whereby PII proteins control the activities of their targets
depends on structural information on the specific complexes
combined with detailed information on the physiological
conditions under which complex formation occurs. To date
such information is relatively limited. However, the solutions
of the AmtB–GlnK [10], PII –NAGK (N-acetyl-L-glutamate
kinase) [24] and PII –PipX [30] structures offer new insights
into the properties of PII proteins that make them so adaptable
for interaction with multiple targets.
Structures of PII proteins complexed with
targets
In nearly all prokaryotes, the amtB gene is co-transcribed
with glnK, strongly suggesting that these two proteins
function together [19]. Evidence for this interaction was
obtained initially from studies in E. coli [20–22] and the
structure of the complex was solved in 2007 [10,23]. In
previous structures of the isolated PII protein, the T-loop
was often unresolved, reflecting its flexible nature, or if it was
resolved, then the observed conformation was constrained
by crystal contacts and hence did not necessarily reflect a
structure that the T-loop might adopt naturally. In the AmtB–
GlnK complex, the T-loops of GlnK adopt a previously
undescribed extended structure with the conserved residue
Arg47 at the loop apex. Each of the T-loops inserts deeply into
the cytoplasmic pore exit of the AmtB conduction channel,
thereby blocking ammonia conduction (Figure 3A). Another
key feature of the complex was the presence of ADP in the
nucleotide-binding site of GlnK. No nucleotide had been
added to the crystallization and the complex had been purified
intact directly from ammonium-shocked E. coli cells. Hence
it was concluded that the presence of ADP was likely to
reflect a physiologically relevant state, and that ADP might
play an important role in influencing the T-loop structure and
promoting complex formation [10,23].
In recent studies, we have monitored in vivo levels of
PII effectors in E. coli during association and dissociation
of the AmtB–GlnK complex. When GlnK is not complexed
to AmtB, intracellular levels of 2-OG and ATP are relatively
high [11]. Consequently, the structure of A. brasilense GlnZ
with bound 2-OG and Mg-ATP (Figure 2C) is probably a
reasonable model for the status of cytoplasmic E. coli GlnK.
When cells become nitrogen-sufficient, intracellular 2-OG
levels fall significantly and there is a transient rise in the ADP
pool [11]. Furthermore, in vitro studies show that binding
of ADP to GlnK is critical for complex formation with
AmtB. Comparison of the structure of the GlnK nucleotidebinding site in the AmtB–GlnK complex with that of GlnZ
reveals that, in the absence of 2-OG, Gln39 relocates to form
a new bond to Lys58 (Figure 2A), thereby influencing the
conformational space occupied by the T-loop. Hence the
switch from bound 2-OG and ATP to bound ADP is likely
to be the key event in promoting AmtB–GlnK complex
formation. In summary, the location of the 2-OG-binding
site near the base of the T-loop offers a likely explanation as
to how 2-OG sensing by PII proteins might be translated into
alternative conformations of the T-loop.
The second structure of a PII complex solved recently
is that of PII with the enzyme NAGK. This interaction
occurs in both cyanobacteria and plants, and structures of the
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Figure 3 Different modes in which PII can interact with its targets
Structures of the complexes of (A) AmtB–GlnK; (B) PII –NAGK; and (C) PII –PipX. In all cases, the T-loops form the major
contacts with the target protein. In AmtB–GlnK, they adopt an extended conformation and infiltrate the ammonia conduction
channels; in PII –NAGK, they are more compact and both the B- and T-loops make contacts with the surface of NAGK; and in
PII –PipX, the T-loops are again extended and form a ‘cage’ that wraps around the ‘tudor’ domains of three PipX molecules
(the C-terminal α-helices of PipX have been omitted for clarity). The Figures were drawn using the software package PyMOL
(DeLano Scientific; http://www.pymol.org) with the PDB codes 2NUU (A), 2V5H (B) and 2XG8 (C).
complex have been solved from both Synechoccocus elongatus
and Arabidopsis thaliana [24,25]. NAGK is the controlling
enzyme of arginine biosynthesis and, in these organisms,
arginine is used as a nitrogen store, thus explaining a role for
PII in controlling NAGK activity. NAGK is a hexamer made
up of two back-to-back homotrimers and, in the complex,
this is sandwiched between two PII trimers (Figure 3B). The
only features in common with the AmtB–GlnK complex are
the symmetry and 1:1 stoichiometry, together with the fact
that the T-loops again constitute a major interface with the
target. Arginine is a feedback inhibitor of NAGK activity,
and the binding of PII rearranges both the allosteric arginine
inhibition site and the catalytic centre of NAGK, thereby
decreasing the affinity for arginine and increasing the affinity
for the substrate NAG (N-acetylglutamate). No effector
molecules were present in the S. elongatus complex structure.
Remarkably, for the A. thaliana complex, well-diffracting
crystals were only obtained in the presence of Mg2+ , ADP,
NAG and arginine, but nevertheless Mg-ATP was found to
be bound to PII in the crystal. The authors conclude that it
was likely to have been bound to the PII when it was purified
from E. coli [25]. In both PII –NAGK complexes, a salt bridge
is present between a conserved lysine residue and glutamate
residue at the base of the T-loop (Figure 2B). Hence, as in the
AmtB–GlnK complex, 2-OG binding to PII will predictably
alter the conformation of the T-loop.
The effects of effector molecules on the PII –NAGK
complex have been analysed in a number of studies for both
S. elongatus and A. thaliana [26–29]. For the cyanobacterial
system 2-OG completely abolishes the interaction providing
that Mg-ATP is also present [28,29]. The plant complex
is much more resistant to 2-OG and only shows gradual
inhibition of complex formation at 2-OG concentrations
greater than 1 mM [26,29]. ATP promotes complex formation
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Authors Journal compilation in both systems, but, whereas ADP is a potent inhibitor of
complex formation in S. elongatus, it is not in A. thaliana [29].
This is presumptively due to the very high affinity of AtPII
for ATP such that it is not easily displaced by ADP [25,29].
Hence, it may be that the two systems have evolved somewhat
different responses to cellular physiology, but further studies
on fluctuations in effector pools in both systems and their
correlation with NAGK activity would be very informative.
The third PII complex structure is that of S. elongatus
PII with another of its targets PipX [30]. PipX is a coactivator of the cyanobacterial transcription factor NtcA,
and PII interaction with PipX antagonizes its ability to
activate NtcA [31]. The complex has a 3:3 stoichiometry,
such that one PII trimer binds three molecules of PipX
(Figure 3C). The PII T-loops are in an extended conformation
resembling, but not identical with, that seen in the AmtB–
GlnK complex and PipX is ‘caged’ between them. Although
no effector molecules are bound in this structure, ADP is
known to increase the affinity of PII for PipX [30]. It is
proposed that binding of 2-OG to PII antagonizes ADP
binding and triggers conformational changes in the T-loops
that facilitate the release of PipX [30]. Such a proposal is
entirely consistent with the role of NtcA in activating gene
expression in response to nitrogen limitation. Hence, as
S. elongatus becomes progressively nitrogen-starved, cellular
2-OG levels are predicted to rise. Binding of 2-OG and
ATP to PII would modify the T-loop conformation, thereby
ensuring that PipX is released from the interaction with PII
and becomes free to activate NtcA.
Conclusions
Studies of PII proteins from a wide variety of organisms have
confirmed that 2-OG, Mg2+ , ATP and ADP are the key
Enzymology and Ecology of the Nitrogen Cycle
effector molecules that regulate the abilities of PII proteins to
interact with their targets. Recent structures of PII proteins
have identified the effector-binding sites and, together with
multiple sequence alignments, these strongly suggest that the
mode of binding for each of the effectors is conserved. It is
now apparent that the interaction of 2-OG with PII is the key
indicator of cellular nitrogen limitation, but that ATP and
ADP also play an important role in regulating interactions
between PII proteins and their targets [11,14]. However, the
precise role of ATP and ADP as regulators of PII interactions
requires further study, and the concept that PII proteins have
the capacity to sense adenylate energy charge [3,9] has yet to
be fully validated.
From the structural data available at present, it is clear
that the T-loops play a key role in most interactions of PII
proteins with their targets. Their conformational flexibility
means that they are able to mediate the interaction with targets
that themselves have widely different structures (Figure 3).
We can also now begin to understand how effector binding
can influence the T-loop conformation and hence the role that
effectors have in the signal transduction process. However, it
should also be noted that the apparent ability of PII proteins to
mediate the formation of ternary protein complexes, in which
the AmtB–GlnK complex recruits a third partner [18,32],
suggests that not all PII interactions will necessarily involve
the T-loops. To date, we only have one system, AmtB–GlnK,
where we are approaching a holistic description of the PII mediated signal transduction process. Consequently, there is a
definite need for the solution of new structures of PII proteins
bound to other targets and for more physiological studies that
can link cellular metabolism to underlying changes in effector
pools.
Funding
This work was supported by the Biotechnology and Biological
Sciences Research Council (BBSRC) [grant number BB/E022308/1
(to M.M.)].
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Received 31 August 2010
doi:10.1042/BST0390189