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J. Mol. Microbiol. Biotechnol. (2000) 2(2): 191-194.
JMMB Correspondence
The ATP-Cone 191
The ATP-Cone: An Evolutionarily Mobile,
ATP-Binding Regulatory Domain
L. Aravind, Yuri I. Wolf and Eugene V. Koonin*
National Center for Biotechnology Information,
National Library of Medicine, National Institutes of Health,
Bethesda, MD 20894, USA
Allosteric regulation is a common mechanism by which
the activity of enzymes is modulated by the concentrations
of their products, substrates and other small regulatory
molecules. Structural studies have suggested that these
functions frequently reside in compact globular domains
that are distinct from the catalytic domains of the enzymes
and are terminally fused to them (Mattevi et al., 1996).
Computational sequence analysis of several domains
involved in allosteric regulation of enzymes have revealed
that they are evolutionarily mobile and the same domain
may be found in a wide range of enzymes that have
unrelated catalytic domains as well as non-enzymatic
proteins, such as membrane transporters and transcription
regulators (Bateman, 1997; Ponting, 1997; Aravind and
Koonin, 1999; Taylor and Zhulin, 1999; Aravind and Koonin,
2000). This distribution pattern suggests that allosteric
regulation primarily evolved as a result of these ligandbinding domains being juxtaposed with different catalytic
domains. Fusion of these ligand-binding domains with
prokaryotic DNA-binding domains, such as HTH, suggests
a common evolutionary origin for allosteric regulation and
effector-mediated transcription regulation (Aravind and
Koonin, 1999).
The highly conserved ribonucleotide reductases are
the paradigm for allosteric regulation of enzymes (Jordan
and Reichard, 1998). Based on biochemical properties,
ribonucleotide reductases fall into 3 classes, namely class
I - B12-independent enzymes, class II- B12-dependent
enzymes, and class III – anaerobic, S-adenosylmethioninedependent enzymes (Jordan and Reichard, 1998). The
class I and class III enzymes from Escherichia coli (NrdA
and NrdD) have been studied in detail, and it has been
shown that their activity is inhibited by dATP and activated
by ATP (Jordan and Reichard, 1998). The crystal structure
of the class I enzyme (NrdA) from E.coli revealed that this
allosteric regulatory site maps to a distinct N-terminal region
(Eriksson et al., 1997) that is also conserved in the
anaerobic class III enzyme (NrdD), but is lacking in the
paralogous class IB enzyme (NrdE) (Eliasson et al., 1996).
This region is conserved in a number of ribonucleotide
reductases of all three classes from different organisms
that have otherwise diverged in the sequence of their
catalytic domains (Tauer and Benner, 1997; Logan et al.,
1999).
Received January 28, 2000; accepted January 28, 2000.
*For correspondence. Email [email protected]; Tel. (301)435-5913;
Fax. (301)480-9241.
© 2000 Horizon Scientific Press
In order to investigate the provenance of this N-terminal
region, we undertook a systematic, iterative search of the
non-redundant (NR) protein sequence database (National
Center for Biotechnology Information, NIH, Bethesda) using
the PSI-BLAST program (Altschul et al., 1997) with a profileinclusion e-value cut-off of 0.01 and the conserved Nterminal domains from different ribonucleotide reductases
as the queries. These searches showed that, in addition
to the well-established relationship among ribonucleotide
reductases, the sequence of this domain is statistically
significantly similar to 2-phosphoglycerate kinases from
archaea and the bacterium Deinococcus radiodurans and
a conserved family of small bacterial proteins typified by
E. coli YbaD. Reverse searches with the regions detected
in the 2-phosphoglycerate kinases and the YbaD-like
proteins retrieved the ribonucleotide reductase N-terminal
regions from the database with e-values<0.001 on
detection over the cut-off. The ribonucleotide reductases
from Methanobacterium, Methanococcus and Chlamydia
and the 2-phosphoglycerate kinase from D. radiodurans
were found to have two-three tandem copies of the
conserved sequence. This allowed precise mapping of the
boundaries of this approximately 85-90 residue region, and
a comparison with the available structure of NrdA (Eriksson
et al., 1997) showed that this exactly corresponds to a
compact, globular N-terminal ATP-binding domain which
includes four α-helices and three ß-strands.
A multiple alignment of the delineated domain was
generated using the conserved regions parsed from the
PSI-BLAST output and adjusted based on the constraints
of the three-dimensional structure of NrdA (Figure 1). The
structure of this domain resembles a cone with the surface
formed by the four helices and the top by a three-stranded
ß-sheet (Figure 2). ATP is bound at the top of the cone just
below the ß-sheet; we named this domain the ATP-cone.
A comparison of the sequence conservation pattern (Figure
1) with the three-dimensional structure (Figure 2) shows
that most of the conserved residues are involved in
stabilizing the secondary structure elements, which
indicates that all these sequences adopt the same
structure. Of interest are the four conserved positions
associated with the N-terminal strands and the first helix
that form hydrogen bonds with the ATP moiety and provide
hydrophobic or stacking contacts (Figure 1,2). The
conservation of these residues leads to the prediction that
most ATP-cone domains bind ATP. The C-terminal helix of
the ATP-cone in the NrdA structure is adjacent to the
catalytic domain and its movements triggered by ligandbinding are likely to mediate allosteric regulation.
The archaeal and deinococcal 2-PGKs catalyze the
ATP dependent phosphorylation of 2-phosphoglycerate
necessary for the synthesis of an unusual metabolite 2-3
cyclic diphosphoglycerate that has been implicated in
denaturation- and thermo-protection of proteins
(Lehmacher et al., 1990; Lehmacher and Hensel, 1994;
Hensel and König, 1998). This enzyme consists of a Ploop-containing kinase domain combined with the N-
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192 Aravind et al.
Figure 1. Multiple alignment of the ATP-cone domain.
The multiple alignment was generated by parsing the HSPs from the PSI-BLAST search outputs and aligning them with structure of the E. coli NrdA (3R1R)
as a guide. The secondary structure shown above the alignment is the consensus derived from the outputs of the prediction programs PSI-PRED and PHD
compared with NrdA structure. The conserved residues that could participate in ATP-binding are shown by asterisks. The 85% consensus shown below the
alignment was derived using the following amino acid classes: polar (p: KRHEDQNST); hydrophobic (h: ALICVMYFW); the aliphatic subset of these are
(l:ALIVMC); big (b:LIFMYWRQE). The limits of the domains are indicated by the position numbers on each side. The sequences are denoted by their gene
name followed by species abbreviation and GenBank Identifier. The species abbreviations are : Hs- Homo sapiens, Sc -Saccharomyces cerevisiae, AtArabidopsis thaliana, VV- Vaccinia virus, T4- bacteriophage T4, MNPV- Mamestra nuclear polyhedrosis virus, Af - Archaeoglobus fulgidus, MtaMethanobacterium thermoautotrophicum, Mj- Methanococcus jannaschii, Ph- Pyrococcus horikoshii, Aae - Aquifex aeolicus, Bs- Bacillus subtilis, Ct- Chlamydia
trachomatis, Dr- Deinococcus radioduran , Ec- Escherischia coli, Mtu- Mycobacterium tuberculosis, Ssp- Synechocystis species, Tma- Thermotoga maritima.
terminal ATP-cone domain, which suggests allosteric
regulation of the catalytic domain by ligands (Figure 3).
Since ATP is one of the substrates of 2-PGK and, at the
same time, one of the products of glycolysis, of which 2phosphoglycerate is an intermediate, it is likely that 2-PGK
is allosterically regulated by ATP.
In addition to the two families of enzymes, the ATPcone domain is found in a set of conserved proteins, typified
by YbaD, that are encoded by most bacteria with large
genomes and Chlamydiae. In these proteins, ATP-cone is
combined with an N-terminal Zn-ribbon domain that could
be involved in nucleic acid-binding (Figure 3). In
Streptomyces, the gene encoding this protein is in the same
operon as the gene for the Class II B12-dependent
ribonucleotide reductase (Genbank gis:3402267 and
3036873), suggesting that it could regulate expression of
ribonucleotide reductase at the level of transcription,
dependent on the binding of ATP or, perhaps more likely,
dATP. Physiologically, it would make perfect sense if, at
high concentrations of dATP, the charged YbaD-family
protein functioned as a transcription repressor, whereas
at low concentrations of dATP, the free form of the regulator
dissociated from the DNA, resulting in transcriptional
activation of the ribonucleotide reductase gene. Although
the operonic association seen in Streptomyces is not
conserved in other bacteria, the strong conservation of the
protein itself suggests that a similar regulatory mechanism
could function in all of them.
Methanobacterium, Archaeoglobus and some nuclear
polyhedrosis viruses encode small proteins that comprise
of an ATP-cone alone (Figures 1, 3). It seems likely that
these proteins also function as regulators of nucleotide
metabolism in a ligand-dependent fashion.
The presence of the conserved ATP-cone domain in
ribonucleotide reductases of all three classes is of particular
interest given the extreme divergence of the catalytic
domain between class I+II and class III (Figure 3). In
principle, this could indicate that the catalytic domain and
the ATP-cone domain have evolved with very different
rates, but it does not seem particularly likely that the
allosteric regulatory domain is so dramatically more
conserved than the enzymatic domain. Rather, the
evolutionary mobility of the ATP-cone (Figure 3) suggests
independent acquisition of this domains by the class I+II
and class III ribonucleotide reductases. In this context, it is
notable that ribonucleotide reductases of archaeal
methanogens contain two copies of the ATP-cone whereas
the Chlamydial enzyme has three copies, emphasizing the
plasticity of the domain architecture (Figure 3). Interestingly,
the catalytic domain and the copy of the ATP-cone domain
adjacent to it in Chlamydia seem to have an eukaryotic
origin (Wolf et al., 1999) whereas the two proximal copies
are most similar to bacterial counterparts (although in this
case, the small size of the domain precludes a conclusive
phylogenetic analysis). Hence it is possible that the
chlamydial ribonucleotide reductase gene arose via
recombination between eukaryotic and bacterial genes,
resulting in a protein with multiple ATP-cone domains. The
multiple ATP-cones could confer allosteric fine-tuning to
their respective catalytic domains in response to different
concentrations of ATP.
The ATP-Cone 193
Figure 2. Structure of the ATP-cone domain
The ribbon diagram of the NrdA (3R1R) ATP-cone domain was produced using MOLSCRIPT. The conserved residues that participate in ATP contacts and
are indicated by asterisks in Figure 1 are shown in a ball-and-stick representation along with the ATP molecule. The hydrogen bonds formed by these
residues with ATP are indicated by dashed lines.
194 Aravind et al.
Figure 3. Domain architectures of the ATP-cone-containing proteins
The various domain architectures of the different ATP-cone domains are depicted along with their phyletic distribution in completely-sequenced genomes.
The species abbreviations are as in Figure 1. The ribonucleotide reductases that lack the ATP-cone are not shown. Zn- stands for the four-cysteine,
rubredoxin-like Zn-ribbon module.
We describe here the wide-spread distribution of the
N-terminal domain of ribonucleotide reductases and show
that it is a prototype of a distinct ATP-binding fold that is
predicted to perform regulatory functions in different
contexts. The predicted allosteric regulation of 2-PGK could
be crucial in certain microbes for denaturation resistance.
The identification of the ATP-cone domain combined with
a Zn-ribbon in the YbaD-family proteins predicts an
unknown but physiologically plausible mechanism of
transcriptional regulation of ribonucleotide reductase
expression in bacteria. The ATP-cone domain joins the
growing class of evolutionarily mobile, ligand-binding
regulatory domains.
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