होम Trends in Microbiology PII signal transduction proteins

PII signal transduction proteins

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15 Diep, D.B. et al. (1998) Secretion and properties of the large and
small lobes of the channel-forming toxin aerolysin. Mol.
Microbiol. 30, 341–352
16 Quiocho, F.A. (1986) Carbohydrate-binding proteins: tertiary
structures and protein–sugar interactions. Annu. Rev. Biochem.
55, 287–315
17 Rudd, P.M. et al. (1997) The glycosylation of the complement
regulatory protein, human erythrocyte CD59. J. Biol. Chem. 272,
7229–7244
18 Abrami, L. et al. (1998) The pore-forming toxin proaerolysin
is processed by furin. J. Biol. Chem. 273, 32656–32661
19 Abrami, L. and van der Goot, F.G. (1999) Plasma membrane
microdomains act as concentration platforms to facilitate
intoxication by aerolysin. J. Cell Biol. 147, 175–184
20 Harder, T. and Simons, K. (1997) Caveolae, DIGs and the
dynamics of sphingolipid cholesterol microdomains. Curr. Opin.
Cell Biol. 9, 534–542
21 Lesieur, C. et al. (1997) Membrane insertion: the strategy of
toxins. Mol. Membr. Biol. 14, 45–64
22 Brown, R.E. (1998) Sphingolipid organization: what physical
studies of model membranes reveal. J. Cell Sci. 111, 1–9
23 Song, L. et al. (1996) Structure of staphylococcal a-hemolysin,
a heptameric transmembrane pore. Science 274, 1859–1866

24 Krause, K.H. et al. (1998) Aerolysin induces G-protein activation
and Ca21 release from intracellular stores in human granulocytes.
J. Biol. Chem. 273, 18122–18129
25 Nelson, K.L. et al. (1999) Channels formed by subnanomolar
concentrations of the toxin aerolysin trigger apoptosis of T
lymphomas. Cellular Microbiol. 1, 69–74
26 Fivaz, M. et al. (1999) Landing on lipid rafts. Trends Cell Biol. 9,
212–213
27 Tweten, R.K. (1995) Pore-forming toxins of Gram-positive
bacteria. In Virulence Mechanisms of Bacterial Pathogens
(Roth, J.A. et al., eds), pp. 207–229, ASM
28 Yamaji, A. et al. (1998) Lysenin, a novel sphingomyelin-specific
binding protein. J. Biol. Chem. 273, 5300–5306
29 Zitzer, A. et al. (1999) Oligomerization of Vibrio cholerae
cytolysin yields a pentameric pore and has a dual specificity for
cholesterol ; and sphingolipids in the target membrane. J. Biol.
Chem. 274, 1375–1380
30 Baorto, D.M. et al. (1997) Survival of FimH-expressing
enterobacteria in macrophages relies on glycolipid traffic. Nature
389, 636–639
31 Parton, R.G. and Lindsay, M. (1999) Exploitation of major
histocompatibility complex class I molecules and caveolae by
Simian virus 40. Immunol. Rev. 168, 23–31

PII signal transduction proteins
Alexander J. Ninfa and Mariette R. Atkinson

T

he signal transduction
PII proteins, found in Bacteria, Archaea
In this article, we will dismechanisms responsible
and plants, help coordinate carbon and
cuss the remarkable PII signal
for the regulation of
nitrogen assimilation by regulating the
transduction proteins, which
development in multicellular
activity of signal transduction enzymes in
are among the most widely
organisms probably evolved response to diverse signals. Recent studies distributed signal transduction
from the systems controlling
of bacterial PII proteins have revealed a
proteins in nature, being presmetabolism in microorganisms.
solution to the signal transduction
ent in Eukarya, Bacteria and
In microorganisms, developproblem of how to coordinate multiple
Archaea. In Bacteria and Arment and metabolism are receptors in response to diverse stimuli yet chaea, PII proteins serve as the
linked at multiple levels. Alpermit selective control of these receptors
central processing unit (CPU)
though they are still largely
under various conditions and allow
for the integration of antagouncharacterized, global reguadaptation of the system as a whole to
nistic signals of carbon and
latory mechanisms couple flux
long-term stimulation.
nitrogen status, and use this
through different branches of
information to control nitroA.J. Ninfa* and M.R. Atkinson are in the Dept of
metabolism to provide overall
gen assimilation. In microrBiological Chemistry, University of Michigan
metabolic coordination. These
ganisms, nitrogen regulation
Medical School, Ann Arbor, MI 48109-0606, USA.
global mechanisms must be
occurs under all growth condi*tel: 11 734 763 8065,
operative even when cells are
tions. Under favorable condifax: 11 734 763 4581,
e-mail: aninfa@umich.edu
growing optimally in preferred
tions, the nitrogen assimilamedium, as indicated by the
tory capacity of the cell is
restraint of various metabolic capacities under these tightly controlled to coordinate nitrogen assimilation
conditions. Metabolic stress, such as starvation for an and carbon assimilation. The response to nitrogen
essential nutrient, results in a variety of responses rang- starvation involves, as a first step, a minor
ing from minor alterations in the cell’s proteome to de- alteration in the cell’s enzymatic capacity. Severe starvelopment of a new cell type or spore. Indeed, bacteria vation leads to the initiation of development in many
of the genus Myxococcus form a multicellular tissue as microorganisms, with the formation of a specialized
part of development triggered by metabolic stress1. cell type that is able to fix atmospheric nitrogen, in
Thus, in microorganisms, development can be thought some cases in symbiosis with plants. Thus, the
of as a very elaborate form of metabolic regulation.
response to nitrogen starvation in microorganisms
0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
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should be thought of as a developmental cascade. Indeed, in many bacteria, the regulatory principles are
similar to the basic Escherichia coli system we will
discuss in detail, with the primary difference being the
degree of specialization of the developed cell type. An
important difference from many other types of development is the reversibility of the development of specialized nitrogen-fixing cell types. As the development of specialized cells is metabolically expensive,
the reversibility of development imposes its own set
of signal transduction requirements, such as ensuring
that transient alterations in the environment do not
trigger a massive waste of energy by causing the cell to
flip back and forth between regulatory states.
The principal short-term signal transduction problems the cell faces in regulating such a developmental
pathway are how to assess the signals (sensation) and
coordinate the function of various signal transduction regulatory proteins to produce a coherent response. Figure 1 shows the roles that PII proteins can
play in this process; this figure is a compilation of the
roles of PII proteins in many organisms and does not
represent the situation in any single organism. PII is
the direct and indirect sensor of various stimuli that
cause the formation of different PII conformations,
each with regulatory significance. The cell contains a
set of PII receptors, which are signal transduction
enzymes that control the activity of transcription factors or key metabolic enzymes. The activity of these
receptors is coordinated by their regulation by PII;
additionally, they could be be subject to independent
regulation in response to signals. A given PII conformation, which indicates a particular cellular state,
will activate or inhibit receptor activities as appropriate, or potentiate the regulation of the receptors by
other factors. In principle, PII could have a variety of
conformations, and the interaction of PII with each
receptor could be optimized to provide precise, rheostat-like control under a variety of cellular conditions. One of the PII receptors can control the expression of a second PII-like protein, which might have
different sensory properties from PII, and/or might
act upon a different set of receptors.
Nitrogen regulation in E. coli and related bacteria
Under favorable growth conditions, such as when
ammonia is present, the assimilation of ammonia into
glutamine is regulated in response to the intracellular
concentrations of glutamine (nitrogen signal) and
2-ketoglutarate (2KG) (carbon signal)2. Under these
conditions, the precise regulation of the activity of
glutamine synthetase (GS), responsible for most of
the assimilation of ammonia, limits the rate of nitrogen assimilation to keep it in balance with the rate of
carbon assimilation. In E. coli and many other bacteria,
this precise regulation of GS is exerted by regulating
expression of its structural gene, glnA, and regulating
GS activity by reversible covalent adenylylation (Fig. 2).
In E. coli, the adenylylation state of GS is controlled
by the bifunctional enzyme adenylyltransferase
(ATase)3, and the transcription of nitrogen-regulated
genes (including glnA) requires the phosphorylated

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Signals

PII

R1

R2

PII-2

R4

R3

Signals

R5
trends in Microbiology

Fig. 1. Incorporation of PII signal transduction proteins into
signal transduction pathways. The figure is a composite illustrating roles that PII proteins can play in different organisms,
and does not correspond to the situation in any single organism. Signals refers to small molecules that control the activity
of the indicated proteins. These can be the same or different
signals for the different proteins. R1, R2, R3, R4 and R5
refer to signal transduction enzymes under the control of PII
or PII-2. As depicted, PII controls R1, R2, and R3 and PII-2
controls R1, R2, R4 and R5. R1 is an enzyme that controls
the activity of a transcription factor that controls the expression
of the structural gene for PII-2.

form of the transcription factor NRI (also known as
NtrC)4 (Fig. 2). The phosphorylation state of NRI
is regulated by the kinase/phosphatase NRII (also
known as NtrB)4. By concerted regulation of NRII
and ATase, the cell can precisely regulate both the
level of GS and its activity. Both NRII and ATase are
controlled, at least in part, by PII (Fig. 2). PII inhibits
the autophosphorylation of NRII and activates its
phosphatase activity5; this results in a rapid decrease
in the extent of NRI phosphorylation (Fig. 2). Upon
binding to ATase, PII activates GS adenylylation
(Fig. 2)6.
PII activity in E. coli is regulated by small-molecule
signals of carbon and nitrogen status. The nitrogen
signal, glutamine, is sensed by ATase and a bifunctional signal transducing uridylyltransferase/uridylylremoving enzyme, UTase/UR (Ref. 7), which controls
PII activity (Fig. 2). The UTase/UR uridylylates PII
when the concentration of glutamine is low, and
deuridylylates PII when the concentration of glutamine is high. As the concentration of PII is fixed,

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NRII is free to phosphorylate
NRI, turning on nitrogen-reguPPi
ATP
lated (Ntr) gene expression.
PII~UMP binds to ATase and
stimulates the deadenylylation of
GS~AMP (Ref. 10), activating GS
ATase
PII~UMP
PPi
H2O
(Fig. 2).
UR
UTase
Gln
GS~AMP GS
Gln
Carbon signals regulate the
ATase
activity
of E. coli PII by directly
PII
UTP
UMP
binding to it (Figs 2,3)7,9,11. The
High 2KG
main carbon signal appears to be
2KG. The trimeric PII contains
ADP
Pi
three binding sites for 2KG and
NRI~P
ATP
NRII
H2O
three binding sites for ATP. The
Gln
NRII
binding of ATP and 2KG to PII
is synergistic. When physiologiPi
NRI
ADP
NRII~P
cal 2KG concentrations are used,
three molecules of ATP bind
Acetate
tightly to PII (Ref. 7). By contrends in Microbiology
Acetyl~P
trast, when ATP is at physiological levels, the first molecule of
Fig. 2 Signal transduction system regulating the activity of glutamine synthetase in Escherichia
coli. Reactions are indicated by curved arrows. The positive and negative influence of effectors and
2KG binds to PII at concenfactors are indicated by straight lines with arrowheads or blunt heads, respectively. Abbreviations:
trations well below the in vivo
ATase, adenylyltransferase; Gln, glutamine; GS, glutamine synthetase; 2KG, 2-ketoglutarate, UR,
level (0.1–0.9 mM)2. This binduridyl removing; UTase, uridylyltransferase.
ing exerts strong negative cooperativity on the binding of additional molecules of 2KG, such
No interaction
that PII only becomes saturated
with ATase or
with 2KG at concentrations apNRII
proaching the high end of the
physiological range (Fig. 3)7,9.
Uridylylation reduces
The binding of 2KG regulates
Kd ~5 µM
negative cooperativity
2KG
the ability of PII to interact
in 2KG binding
with ATase and NRII, with
P
Low Gln
the fully saturated form being
UM
unable to interact with either
Interacts with
protein7,9,11–13. Thus, the carbon
ATase and NRII
High Gln
signal can counteract the effect of
the nitrogen signal, resulting in
an integration of the two types of
High Gln
Low Gln
information (Fig. 3). The carbon
Kd ~150 µM
and nitrogen signals are sensed
independently, as PII with variUM
P
ous amounts of bound 2KG
No interaction
P
UM
can be acted upon by UTase/UR
with ATase or
Low Gln
No interaction with
(Fig. 3).
NRII
NRII, interacts with
We are only beginning to unHigh Gln
ATase
derstand some of the more subtle
aspects of the signal transduction
trends in Microbiology
system. For example, in vitro
experiments indicate that glutaFig. 3. Regulation of PII conformation. The main carbon signal is 2-ketoglutarate (2KG). Trimeric PII
mate and oxaloacetate also interhas three 2KG-binding sites and three uridylylation sites. The binding of 2KG influences the ability
of PII to interact with adenylyltransferase (ATase) and a kinase/phosphatase (NRII), both of which
act with PII, and do so by binding
are involved in nitrogen regulation. At low 2KG concentrations, the conformation of PII is such that it
to the 2KG-binding site9. These
is able to interact with ATase and NRII. At high 2KG concentrations, the conformation of PII is such
effectors
bind PII much less well
that it cannot interact with ATase and NRII. Additionally, uridylylation reduces the negative cooperthan
2KG
does. In vitro, glutaativity in 2KG binding. Ovals, circles and triangles are used to represent three different conformations
of PII. Small black dots are used to represent bound molecules of 2KG.
mate binding can result in a unique
PII conformation that interacts
with ATase and NRII, but not
uridylylation of PII regulates the concentration of with UTase/UR (Refs 9,11,13). It is possible that, in
both PII and PII~UMP. PII~UMP does not bind to cells, the activity of PII is modulated by the concerted
NRII (Refs 8,9), and thus when glutamine is low, effects of several carbon signals (Fig. 1).
Gln

UMP

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A key point about the signal transduction system
in E. coli is that the concentration of the components
must be balanced in order for the signaling mechanism to function properly – the system is fine-tuned.
PII, UTase/UR and ATase are constitutively expressed
at low levels14. Overexpression of PII from multicopy
plasmids renders the system unable to respond to
nitrogen starvation, whereas underexpression of PII
results in the starvation response in the absence of
starvation, as expected (A.J. Ninfa and M.R. Atkinson,
unpublished). Similarly, overexpression of the PII
receptors NRII or ATase from multicopy plasmids
renders regulation by PII irrelevant, as expected
(A.J. Ninfa and M.R. Atkinson, unpublished). The
cell might use this property of the system to limit
regulation by PII to conditions that are not stressful.
A part of the response to nitrogen stress is an increase
in the intracellular concentrations of NRI and NRII
(Fig. 4)15,16. The main role of PII could be to prevent
this amplification in the levels of NRII and NRI
occurring when the cells are not nitrogen starved.
Severe nitrogen starvation, causing an amplification
of NRII and NRI, results in a staged response to adverse conditions. At the second level of the cascade
(Fig. 4), Ntr genes are expressed, the products of which
might permit the uptake or catabolism of alternative
nitrogen sources, or the more efficient transport of
ammonia. Additionally, in related bacteria, regulatory
proteins are expressed at the second level that control
the expression of genes at a third level (Fig. 4)17–20. In
some nitrogen-fixing organisms, the components of
the nitrogen-fixing apparatus are encoded by nif
genes at the third level of the cascade.
Understanding the nature of the switch between
the first and second levels of the cascade requires a
brief diversion into the mechanism of transcriptional
control by the phosphorylated form of NRI (NtrC).
This factor binds upstream enhancers21, and interacts
with s54 RNA polymerase at the target promoters by
means of a DNA loop22. The formation or stability of
the DNA loop can be regulated by accessory transcription factors23,24. Different Ntr promoters require
different levels of NRI~P for activation17,18. This can
result from several factors, such as enhancers of various strength, and promoters with various affinities
for s54 RNA polymerase. The net effect is that each
Ntr promoter is ‘hardwired’ to become activated only
when the NRI~P concentration is above a certain
threshold. Negative regulation by NRI~P has also
been observed in vitro and in vivo18,25, presumably
caused by NRI~P binding at silencer sites that, for example, prevent the formation of the activating DNA
loop. Thus, the expression of glnA first increases and
then decreases as the concentration of NRI~P is increased18,25. The Ntr genes at the second level of the
cascade in Fig. 4 have promoters that require a higher
level of NRI~P for activation than does the glnA
promoter.
One of the genes expressed at the second level of
the cascade in E. coli and its close relatives is glnK,
which encodes GlnK (Ref. 19), a PII-like protein
(Fig. 4). GlnK appears to be specifically designed to

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glnALG

GS

NRI

Level 1

NRII

PII

NRII

NRI~P
Signals

Ntr genes

nifLA

glnK

Level 2
GlnK

NifA

NifL

nif genes

Level 3
trends in Microbiology

Fig. 4. Developmental cascade leading to the expression of
nitrogenase in Klebsiella pneumoniae. Some aspects of the
pathway were deduced in the closely related Escherichia coli.
Arrowheads indicate activation, or the product of the indicated gene, flattened arrowheads indicate inhibition and the
round arrowhead indicates activation by a low concentration
of NRI~P and inhibition by a high concentration of NRI~P.
Signals refers to small molecules that bind to PII and GlnK,
and regulate their activities. As depicted, GlnK prevents NifL
from inhibiting NifA. However, it is not certain that GlnK interacts
with NifL as opposed to NifA; GlnK might bind NifA and prevent inhibition by NifL. Abbreviation: GS, glutamine synthetase.

function under conditions of nitrogen starvation.
GlnK can interact with and regulate NRII and ATase,
although it is much less effective in regulating ATase
than is PII (Refs 20,26). Also, like PII, GlnK can be
modified by UTase/UR and can bind 2KG and ATP,
although the binding to 2KG has slightly different
properties26. The most obvious difference between
GlnK and PII is that, unlike PII~UMP, GlnK~UMP is
not readily deuridylylated by UTase/UR (Ref. 26).
The role of GlnK in E. coli is not understood, as the
null mutant has no obvious phenotype in otherwise
wild-type cells. In principle, GlnK might provide better control of NRII and ATase under stressful conditions. For example, it has been proposed that GlnK
could serve as a ‘memory protein’ that prevents the
inappropriate termination of the stress response by a
transient improvement in the environment19. According to this hypothesis, the cell’s ability to perceive and
act upon stimuli is tempered by its recent history.
A key to understanding one role of GlnK comes
from the observation that GlnK, but not PII, regulates
the activity of NifA or NifL in nitrogen-fixing bacteria such as Klebsiella pneumoniae27,28 (Fig. 4). Thus,
GlnK regulates development by controlling receptors
other than NRII and ATase. It seems likely that additional GlnK receptors will be identified, as there is at
least suggestive evidence for their existence in several
cases.

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Table 1. PII proteinsa
Organism
Bacteria
Escherichia coli

Klebsiella pneumoniae
Haemophilus influenzae
Herbaspirillum seropedicae
Rhizobium meliloti
Bradyrhizobium japonicum
Rhizobium leguminosarum
Rhizobium etli
Azospirillum brasilense

Protein

Sequence accession

PII
GlnK
PII
GlnK
PII
PII
PII
PII
PII
GlnK
PII
Pz
PII
GlnK
PII
PII
PII
GlnK
PII
PII
NrgB
PII
PII
PII
GlnK
PII
PII
PII
PII
PII
PII
PII
PII

spP05826
spP38504
spP11671
embCaa07092.1
spP43795
gi1835631
sp052905
spP14179
spP09827
embCAA05496
spP21193
embCAA63238
embCAA71264
embCAA12409
gi2735324
gi664947
gi2642627, spP43519
gi2565279
spP13556
embCAB39373.1
sp007428
gi100079
g2258162
gbAAD56036.1
gi483084
gbAAD35488
gi2982852
sp010960
sp055247
pirA9696
sp030794
sp047894
embCAA22427

GlnK vary. In addition, it has been
shown that GlnK and PII form
heterotrimers29, which could have
properties differing from either homotrimer. This could permit rheostatlike control of CPU performance.
The ability to vary the CPU continuously implies that both sensation and
system output are optimized with
regard to the physiological state and
prior history of the cell.

PII proteins in other bacteria
Given their distribution in nature
(Table 1), we can expect a priori that
PII proteins have evolved in different
ways to perform different functions
Azorhizobium caulinodans
in various organisms. Several fundaAzotobacter vinelandii
mental differences from the situation
Rhodospirillum rubrum
in E. coli have already been noted.
Rhodobacter sphaeroides
Bacteria differ in the number of PII
proteins, in the location of the genes
Rhodobacter capsulatus
encoding them, and in the regulation
Corynebacterium glutamicum
of the expression of these genes. The
Bacillus subtilis
glnB gene encoding PII can be found
Clostridium longisporum
as a single-gene operon30, as part of
Clostridium cellobioparum
an operon with glnD (encoding
Xanthomonas citri
UTase/UR)31 or as part of an operon
Desulfovibrio gigas
Thermatoga maritima
with glnA (Refs 32,33). In many of
Aquifex aeolicus
the organisms in which glnB is linked
Mycobacterium tuberculosis
to glnA, PII is expressed at a low level
Synechocystis sp.
under conditions of nitrogen excess,
Synechococccus sp.
and both PII and GS become highly
Nostoc punctiforme
expressed under nitrogen-limiting
Fremyella diplosiphon
conditions34. In this arrangement, a
Streptomyces coelicolor
single PII protein can be thought of as
playing both ‘PII’ and ‘GlnK’ funcArchaea
tions, with the fine-tuning of the conMethanobacterium thermoautotrophicum PII
sp026760, sp026758
GlnK?
sp050786
centration of the cellular signal transGlnK?
gi2622683
duction components playing a key
Methanococcus jannaschii
GlnK
sp060381
role in PII function. However, some
Methanobacterium ivanovii
GlnK?
spP51604
organisms with this configuration
Archaeoglobus fulgidus
PII (glnB-1)
gi2649618
have, in addition, a distinct GlnK
GlnK? (glnB-2) gi2648808
(Ref. 33). In other cases, the glnAGlnK? (glnB-3) gi2648801
linked gene seems to encode a bona
fide GlnK-like protein involved in the
Eukarya
control of NifA activity (and not inArabidopsis thaliana
PII
gi3885943
volved in the regulation of GS under
Ricinus communis
PII
gi3885941
Porphyra purpurea
PII
spP51254
non-stressful conditions)35.
Several lines of evidence suggest
a
PII proteins were identified by performing a basic BLAST search of the non-redundant transthe existence of additional PII/GlnK
lation of the DNA sequence library at the National Center for Biotechnology Information (NCBI)
receptors. In methanogens, there are
web site (http://www.ncbi.nlm.nih.gov) using the amino acid sequences of E. coli PII,
two GlnK proteins encoded by the nifH
B. subtilis NrgB and X. citri PII as the query sequences.
operon36. These might help regulate
covalent modification of nitrogenase
If PII can be thought of as the CPU that integrates by the DraG/DraT system, or regulate another factor
information and controls receptors when the cell is in downstream of nif expression in the cascade. Bacillus
one physiological state, then GlnK can be thought of subtilis contains a PII-like protein, NrgB, which, like
as reflecting the ability of the cell to modify (or re- E. coli GlnK, is in a nitrogen-regulated operon with a
place) its CPU for better performance under a differ- putative ammonia transport protein37. However,
ent set of conditions. The expression of GlnK is B. subtilis contains neither NRII nor ATase, and thus
tightly controlled, so the relative amounts of PII and NrgB must interact with another receptor.

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In cyanobacteria, PII is not
regulated by reversible covalent
(a)
(b)
uridylylation, but rather is controlled by reversible phosphoryT loop
lation of a serine residue38,39.
The kinase and phosphatase activities, which seem to reside on
distinct enzymes, are regulated
B loop
synergistically by ATP and 2KG,
which bind to PII (Refs 40,41).
These results suggest that the
ability of PII to directly sense
small-molecule effectors pre-dates
C loop
the development of covalent
modification systems to control
PII activity. PII has a variety of
functions in cyanobacteria, including control of nitrate and
Fig. 5. Ribbon cartoon of (a) PII and (b) GlnK. For each protein, a similarly oriented monomer is
nitrite uptake, possibly by direct
shown. The three surface-exposed loops are shown. Sequences in the T loop and B loop are highly
conserved. It can be seen that the structure of PII and GlnK are similar, although the conformation
interaction with a component
of the T loop differs between the two proteins. The figure was constructed using Rasmol 2.6.
of the uptake systems. Nitrate
assimilation is coupled to photosynthesis in these organisms,
with the latter process providing the reducing equiva- proteins are not subject to covalent modification. The
lents necessary for the former. PII regulates the genome sequence of B. subtilis and the available arcoupling of these two physiological processes.
chaeal sequences have not revealed an enzyme related
to E. coli UTase/UR. Of the three eukaryotic PII proPII proteins in Eukarya
teins, that from P. purpurea contains the sequence
There are three reports of PII proteins in Eukarya. A YKGSEY at this position, rendering it a candidate for
PII protein is encoded by the chloroplast genome of either serine or tyrosine modification, or both, whereas
the red algae Porphyra purpurea, and the protein is a the PII proteins from A. thaliana and R. communis
chloroplast protein42. In two higher plants, Arabidopsis contain HGGSEF and QGGSEF at this position,
thaliana and Ricinus communis, a gene encoding a respectively, suggesting that these proteins are not
PII protein is present within the nucleus, and the uridylylated but could be phosphorylated on serine
nuclear-encoded PII protein is transported to the like the cyanobacterial PII.
chloroplast43. The identity of the PII receptors in the
Given the observation that PII and GlnK functions
chloroplast are unknown.
might be divided up in various ways between a cell’s
complement of PII proteins, it is difficult to discern
Structure and function of PII and GlnK proteins
the relatedness of the various GlnK and PII proteins.
Currently, 44 highly related sequences encoding PII Some of the proteins named PII for historical reasons
proteins are known (Table 1). The PII proteins consist are probably actually GlnK and vice versa, whereas
of ~112 amino acids and are highly conserved some proteins might have mixed functions. In any
throughout their entire length, with the archaeal case, for most of the proteins in the database we lack
proteins having an extra block of nine amino acids in a clear idea as to their physiological function. Therethe T loop, as discussed later. Two sequence motifs fore, we will simply note that when a cell contains
are most highly conserved. The first of these more than one PII-like protein, these are often no
(AXTGXIGDGKIF, the sequence in bold entirely con- more closely related than are PII proteins from differserved) is found at position ~81–92 and constitutes ent organisms. For example, E. coli PII and GlnK disthe B loop and flanking regions of the protein. The play 36 differences over their 112 amino acids, rensecond conserved motif (YRGAEY, most conserved dering them less homologous than the PII proteins
residues in bold) at position ~46–51 constitutes the from close relatives of E. coli.
apex of the T loop, and contains the site at which
The structure of E. coli PII in the absence of ligE. coli PII is uridylylated, Y51. In cyanobacteria, the ands has been determined by Ollis and colleagues44,45;
corresponding sequence is YRGSEY, with the site of a cartoon of the monomer structure is shown in
phosphorylation being the S49 residue. A similar se- Fig. 5. The trimer forms a short, squat barrel with
quence (YRGSEI) is found in Clostridium longispo- an inner channel. At the interfaces of the subunits,
rum, suggesting that this protein could also be phos- three surface-exposed loops are found on the outer
phorylated at serine. Yet another sequence is found in edges of the barrel, two of which, the T and B loops,
the Archaeoglobus fulgidus proteins, which have the are from one subunit, with the C loop contributed
sequence FRGREV or FRGRTM at this position. by the adjacent subunit. The B loop is formed by
B. subtilis NrgB has YRGVKI at this position, and the most highly conserved sequence motif. The strucThermatoga maritima has YRGEVE. Perhaps these ture of the trimer suggests that the loops and the

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Questions for the future
• How do the small-molecule effectors of PII control the conformation of the T loop, and what are the biologically important
conformations of the T loop? Where does 2KG bind to PII?
• What receptors does PII interact with (in addition to NRII and
ATase), and what are their physiological roles?
• How does the T loop of PII interact with its receptors? Do other
surfaces of PII also touch the receptors?
• What are the physiological roles of the GlnK-like proteins?

clefts between them serve as sites of regulatory
interactions44.
The Ollis group also solved the structure of unliganded and ATP-bound GlnK (Ref. 46; Fig. 5). As
expected, the structures of PII and GlnK are similar,
permitting the solution of the GlnK structures by
molecular replacement. However, the conformation
of the T loop in the GlnK structure (bound to sulfate)
differs considerably from the conformation of the PII
T loop (Fig. 5). In GlnK, the asymmetric unit contains
two GlnK monomers, one of which has a disordered
T loop, whereas the other T loop is more compact,
containing a short 310 helix47 at its apex. The observation of different T loop conformations in the PII
and GlnK structures available so far suggests that,
in solution, the T loop can adopt several conformations. In the structure of ATP-bound GlnK, the site
of ATP binding is a cleft flanking the B loop at the
base of the T loop. From this position, ATP might influence the conformation of the T loop. Unfortunately, the T loop was disordered in the GlnK–ATP
structure.
The structures available so far have provided a
framework for understanding the properties of mutant forms of the proteins, and provided the basis for
solving additional structures by molecular replacement. However, the structures of the physiologically
important forms of PII and GlnK remain to be determined. These are PII–(ATP)3–2KG; PII–(ATP)3–
(2KG)3; PII–(ATP)3–Glutamate; GlnK–(ATP)3–2KG;
(PII~UMP)3–(ATP)3–(2KG)3, as depicted in Fig. 3.
Solving these structures could reveal biologically
important T loop conformations, and how the T loop
conformation is controlled by effector binding.
Functional analysis of the E. coli PII protein has
shown that the T loop is responsible for interaction
with NRII and ATase, and that some single amino
acid alterations in the T loop result in PII proteins
specifically defective in interaction with each receptor.
Based on the PII structure, a deletion of the apex of
the T loop was designed and the altered protein characterized. This protein displayed normal ATP and
2KG binding, but was unable to interact with NRII,
ATase or UTase/UR (Ref. 48). However, when heterotrimers were formed containing one wild-type PII
subunit and two subunits bearing the deletion, these
heterotrimers were able to regulate NRII and ATase,
and the wild-type subunits in the heterotrimers could
be uridylylated by UTase/UR (Ref. 49). These experiments suggest a single T loop of PII is required to
interact with its receptors.

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The interaction of PII with its receptors requires
the binding of a single molecule of 2KG to the PII
trimer9,11–13 (Fig. 3). The presence of NRII stimulates
the binding of the first molecule of 2KG by PII
(Ref. 9), as one would expect from Fig. 3. Among the
single substitution mutations, the mutation A49P
near the apex of the T loop is of particular interest48.
This mutation specifically abolishes the interaction of
PII with NRII and is predicted to prevent the formation of a T loop 310 helix47, as observed in one of
the GlnK structures46. Another interesting property of
the A49P protein is that it is altered in 2KG binding;
specifically, the negative cooperativity of 2KG binding is diminished48 (Fig. 2). These results suggest
mutual influence of T loop conformation and the
conformation of the 2KG-binding site.
Alterations in the PII B loop result in defects in
ATP binding (in agreement with the GlnK~ATP
structure) and 2KG binding, and in all other PII activities48. Also, certain alterations in the T loop affect
2KG. In particular, the mutation Q39E, located near
the base of the T loop, results in drastically diminished binding of 2KG to PII (Ref. 48). The side chain
of Q39 projects towards the B loop of PII; it is possible that this residue forms part of the 2KG-binding
site. Alternatively, as ATP and 2KG binding are
synergistic, Q39 could form part of the system for
communication between the ATP-binding site (B loop)
and the 2KG-binding site.
Conclusions
The overall picture emerging from studies so far is
that small-molecule-effector binding influences the
conformation of the T loop, which interacts with
receptors. As the binding of receptors also influences
the conformation of the T loop, with attendant effects
on small-molecule-effector binding, the unmodified
form of PII can be thought of as a sensory element
that permits the receptors to perceive various effectors. The different receptors interact with PII in subtly different ways, as indicated by receptor-specificity
mutations in the PII T loop; thus, the binding of
different receptors to PII might result in different
effector-binding properties of the complexed PII.
This could permit sensation to be optimized for each
receptor using a common PII.
Acknowledgement
Research in our laboratory is supported by grants GM47460 and
GM57393 from the NIH.
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