Download CBS domains: structure, function, and pathology in human proteins

Am J Physiol Cell Physiol 289: C1369 –C1378, 2005;
doi:10.1152/ajpcell.00282.2005.
Invited Review
CBS domains: structure, function, and pathology in human proteins
Sofie Ignoul and Jan Eggermont
Laboratory of Physiology, K.U. Leuven, Campus Gasthuisberg O&N, Leuven, Belgium
chloride channel; cystathionine ␤-synthase; AMP-activated protein kinase
PROTEIN DOMAINS are evolutionarily conserved units with shared
structural (sequence and folding pattern) and functional properties. It is generally assumed that each protein domain performs a specific function. However, because an identical domain may occur in proteins with completely different functions
(e.g., channels, kinases, metabolic enzyme), a major challenge
is to relate the domain function to the overall protein function.
A nice example of this conundrum is posed by cystathionine␤-synthase (CBS) domains. These domains were first described in 1997 by Bateman (3), who identified in the genome
of the archaebacterium Methanococcus jannaschii a group of
proteins containing one or more copies of a conserved domain
of ⬃60 amino acids. Homology screening of protein databases
revealed that this domain was widespread, not only in archaebacterial proteins but also in eubacterial and eukaryotic proteins, e.g., human CBS, from which the name of the domain is
derived (3). Moreover, this analysis also indicated that CBS
domains usually come in tandem repeats, which associate to
form a so-called Bateman domain or a CBS pair. An up-to-date
list of proteins with CBS domains is found in the NCBI
Conserved Domain Database (see http://www.ncbi.nlm.
nih.gov/Structure/cdd/cddsrv.cgi?uid⫽pfam00571). Table 1
summarizes the occurrence of CBS domain-containing proteins
in prokarya and eukarya.
Although their precise function remains to be elucidated, the
(patho)physiological importance of CBS domains is emphasized by the observation that point mutations in CBS domains
can seriously cripple the specific protein function and are
Address for reprint requests and other correspondence: J. Eggermont, Laboratory of Physiology, K.U. Leuven, Campus Gasthuisberg O&N, Herestraat 49,
B-3000 Leuven, Belgium (e-mail: [email protected]).
http://www.ajpcell.org
responsible for several hereditary diseases in humans (see also
Fig. 5): retinitis pigmentosa [RP; mutation in inosine-5⬘-monophosphate (IMP) dehydrogenase (IMPDH)] (38), homocystinuria (CBS) (69), familial hypertrophic cardiomyopathy with
Wolff-Parkinson-White syndrome [AMP-activated protein kinase (AMPK)] (4, 22, 23), myotonia congenital (ClC-1) (64),
idiopathic generalized epilepsy (ClC-2) (28), Dent’s disease
(ClC-5) (46), osteopetrosis (ClC-7) (9, 42), and Bartter syndrome (ClC-Kb) (41). In this review, we first focus on the
structure of CBS domains and then discuss the role of these
domains in the function or dysfunction of IMPDH, CBS,
AMPK, and CLC chloride channels.
STRUCTURE OF CBS DOMAINS
The first clue as to the structure of CBS domains came from
the crystal structure of Chinese hamster (Cricetulus griseus)
IMPDH, which contains two CBS domains in tandem (71). On
the basis of these data, it was proposed that a single CBS
domain consists of a conserved ␤1-␣1-␤2-␤3-␣2 pattern (see
Fig. 1). However, the CBS domains appeared relatively disordered in the crystal structure, which precluded a detailed
structural view of the CBS pair, i.e., the overall structure
formed by the association of the two domains. More recently,
the crystal structures of several bacterial proteins containing
one or more CBS domains have been resolved, e.g., IMPDH
from Streptococcus pyogenes (Protein Data Bank code 1ZFJ)
(78) and three functionally uncharacterized proteins from,
respectively, Thermothaga maritima (PDB code 1O50) (53),
Thermoplasma acidophilum (PDB code 1PVM) (see http://
www.rcsb.org/pdb/cgi/explore.cgi?job⫽graphics&pdbId⫽
1PVM&page), and Methanothermobacter thermautotrophicus
(PDB code 1PBJ) (see http://www.rcsb.org/pdb/cgi/explore.
0363-6143/05 $8.00 Copyright © 2005 the American Physiological Society
C1369
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Ignoul, Sofie, and Jan Eggermont. CBS domains: structure, function, and
pathology in human proteins. Am J Physiol Cell Physiol 289: C1369 –C1378, 2005;
doi:10.1152/ajpcell.00282.2005.—The cystathionine-␤-synthase (CBS) domain is
an evolutionarily conserved protein domain that is present in the proteome of
archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem
repeats and are found in cytosolic and membrane proteins performing different
functions (metabolic enzymes, kinases, and channels). Crystallographic studies of
bacterial CBS domains have shown that two CBS domains form an intramolecular
dimeric structure (CBS pair). Several human hereditary diseases (homocystinuria,
retinitis pigmentosa, hypertrophic cardiomyopathy, myotonia congenital, etc.) can
be caused by mutations in CBS domains of, respectively, cystathionine-␤-synthase,
inosine 5⬘-monophosphate dehydrogenase, AMP kinase, and chloride channels.
Despite their clinical relevance, it remains to be established what the precise
function of CBS domains is and how they affect the structural and/or functional
properties of an enzyme, kinase, or channel. Depending on the protein in which
they occur, CBS domains have been proposed to affect multimerization and sorting
of proteins, channel gating, and ligand binding. However, recent experiments
revealing that CBS domains can bind adenosine-containing ligands such ATP,
AMP, or S-adenosylmethionine have led to the hypothesis that CBS domains
function as sensors of intracellular metabolites.
Invited Review
C1370
STRUCTURE AND FUNCTION OF CBS DOMAINS
Table 1. Current distribution of CBS domains
among different phylae
Species
0
281
1,650
113
48
182
50
33
23
42
28
104
12
CBS, cystathione ␤-synthase. Data were drawn from http://www.ebi.ac.uk/
interpro/DisplayIproEntry?ac⫽IPR000644.
cgi?job⫽graphics&pdbId⫽1PBJ&page). These data confirm
the originally proposed ␤1-␣1-␤2-␤3-␣2 pattern for a single
CBS domain, albeit with a cautionary note for the first ␤-strand
(␤1), which is less reliably predicted in some structures (see
Fig. 2A). Moreover, these crystal structures also show how two
CBS domains associate to form a CBS pair (Fig. 2B). In
essence, a CBS pair is a symmetrical structure that is composed
of two (␤1)-␣1-␤2-␤3-␣2 units in an antiparallel arrangement.
The four ␣-helices are positioned at one side of the pair (Fig.
Fig. 2. Structural model of a CBS pair (CBS1 and CBS2). A: Swiss-Pdb
Viewer (http://us.expasy.org/spdbv/) and DS ViewerProwere used to visualize the CBS1 and CBS2 domains of Streptococcus pyogenes IMPDH as
given by the atomic coordinates of the crystallized protein (Protein Data
Bank code 1ZFJ) (78). ␣-Helices are shown in red, ␤-strands in blue. The
two CBS domains, CBS1 (left) and CBS2 (right), have a (␤1)-␣1-␤2-␤3-␣2
structure (in both domains, ␤1 is not clearly resolved as a ␤-sheet). The
CBS pair is formed by an antiparallel association of the CBS domains. The
interface of the 2 CBS domains is formed by a ␤-plate that consists of the
antiparallel ␤2-␤3 sheets of each CBS domain. In the CBS pair, the four
␣-helices are positioned at one end (top), whereas the loops connecting the
␤2-␤3 sheets are at the other end of the structure (bottom). B: the molecular
surface of the CBS pair was constructed using DS ViewerPro and was
based on the atomic coordinates of the CBS pair from the bacterial IMPDH
(PDB code 1ZFJ) (78). Electrostatic potential at the surface is depicted as
blue for positive and red for negative. The interface between the two CBS
domains forms a cleft, which could form a ligand binding site. At the
bottom of this cleft resides an aspartate or asparagine residue that is
conserved in the crystallized bacterial proteins.
Fig. 1. The cystathionine-␤-synthase (CBS) domain: secondary structure. The
conserved ␤1-␣1-␤2-␤3-␣2 pattern of the CBS domain as originally derived
from the crystal structure of hamster inosine-5⬘-monophosphate dehydrogenase (IMPDH) (71) is shown. The ␣-helices are depicted as dark gray
rectangles, whereas the light gray arrows represent the ␤-strands. The ␤1-␣1␤2-␤3-␣2 pattern is conserved among different CBS domains with the exception of the first ␤-strand (␤1) that does not always show up as a clear ␤-sheet
in some of the crystal structures.
AJP-Cell Physiol • VOL
2, top), whereas the loops connecting the antiparallel ␤2-␤3
sheets are located at the other side of the structure (Fig. 2A,
bottom). Molecular surface computation suggests the presence
of a cleft between the two CBS domains, which contains in the
crystallized bacterial proteins a conserved aspartate or asparagine at the bottom of the cleft (see Fig. 2B). It is unknown
whether this paired structure also applies to mammalian CBS
domains. Modeling of the human ClC-1, AMPK, and CBS
domains on the bacterial templates suggests that the human
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Virus
Prokarya
Archaea
Eubacteria
Eukarya
Green plants
Arabidopsis thaliana
Chordata
Homo sapiens
Arthropoda
Drosophila melanogaster
Nematoda
Caenorhabditis elegans
Fungi
Saccharomyces cerevisiae
No. of Proteins With CBS Domains
Invited Review
STRUCTURE AND FUNCTION OF CBS DOMAINS
HUMAN CBS PROTEINS AND ASSOCIATED DISEASES
CBS domains have been identified in a wide range of
proteins, both soluble and membrane proteins. CBS domaincontaining proteins have very divergent functions, ranging
from metabolic enzymes and transcriptional regulators to ion
channels and transporters. Therefore, an intriguing question is
whether CBS domains serve a common function in these
different proteins or whether CBS domains are polyfunctional
units. To discuss this problem, we give an overview of human
(mammalian) CBS domain-containing proteins.
Cystathionine ␤-Synthase
Human CBS performs a crucial step in the biosynthetic
pathway of cysteine by providing a regulatory control point for
S-adenosylmethionine (AdoMet) (19). CBS catalyzes the condensation of L-serine and L-homocysteine, thereby forming
L,L-cystathionine, which is subsequently cleaved to cysteine
(for review, see Ref. 52). Alternatively, L-homocysteine, after
being methylated to methionine, can be converted to AdoMet,
which donates methyl groups to a variety of substrates, e.g.,
neurotransmitters, proteins, and nucleic acids. AdoMet functions as an allosteric activator of CBS by increasing the
enzyme activity about threefold (20). In doing so, AdoMet
exerts control on its biosynthesis: low concentrations of
AdoMet result in low CBS activity, thereby funneling homocysteine in the transmethylation pathway toward AdoMet formation. In contrast, high AdoMet concentrations allow the
clearance of homocysteine into the transsulfuration pathway,
leading to cysteine biosynthesis (see Fig. 3A). It has recently
been proposed (7) that CBS can also generate H2S, a putative
paracrine modulator in the brain and the vascular system, via a
condensation reaction involving cysteine and homocysteine.
Human CBS contains 551 amino acids and forms a homotetrameric enzyme complex in vivo (44) capable of associating
into higher oligomers in solution (43). The COOH-terminal
region of CBS encompasses a CBS domain (CBS1: amino
acids 416 – 468), as originally described by Bateman (3),
whereas sequence alignment with S. pyogenes IMPDH identified a second, less-conserved CBS domain (CBS2: amino acids
486 –543) further down to the COOH terminus (69).
Several observations indicate that the COOH-terminal CBS
domains are directly responsible for AdoMet binding and
activation of enzyme activity. First, removal of the COOHterminal CBS domains either by limited trypsinolysis or by
site-directed mutagenesis generates a truncated CBS that is no
longer activated by AdoMet (39). Consistent with this observation is the finding that Trypanosoma cruzi cystathionine
␤-synthase, which lacks the COOH-terminal CBS domains, is
not activated by AdoMet (61). However, it should be pointed
out that yeast cystathionine ␤-synthase, which has two CBS
domains in its COOH terminus, is not regulated by AdoMet
(47). Second, specific point mutations in the CBS domains of
cystathionine ␤-synthase, e.g., D444N and S466L mutations in
the first CBS domain, result in an AdoMet-insensitive enzyme
(35, 40, 69). Third, the studies of Janosik et al. (35) revealed
that the CBS domains form an autoinhibitory domain in cystathionine ␤-synthase, the effect of which is relieved by
AdoMet binding. Fourth, recent work (68) involving binding
Fig. 3. Model for metabolic sensor function
of CBS domains. A: allosteric activation of
CBS by S-adenosylmethionine (AdoMet) determines the metabolic fate of L-homocysteine. At low cytosolic levels of AdoMet,
CBS is poorly active and L-homocysteine is
funneled into the transmethylation pathway
leading to the synthesis of AdoMet, which
donates methyl groups to various acceptors
(top). In contrast, high AdoMet levels activate CBS, which catalyzes the conversion
of L-homocysteine into L,L-cystathionine, a
precursor of L-cysteine (transsulfuration
pathway; bottom). B: binding of AMP to
AMP-activated protein kinase (AMPK) provides a feedback signal for the cellular energy status. A decrease in cellular energy
levels, as reflected by an increase in the AMP
level activates AMPK, which switches on
catabolic pathways (ATP production) and
switches off anabolic pathways (ATP consumption), thereby raising the cellular energy
level (increase in ATP, decrease in AMP).
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CBS domains can adopt the typical (␤1)-␣1-␤2-␤3-␣2 core
structure and form an intramolecular CBS pair.
It should be noted that in the bacterial proteins the CBS pairs
are always formed by intramolecular association of two CBS
domains. Although at this time one cannot formally exclude
the formation of an intermolecular CBS pair, the presently
available data indicate that the above-described interaction
between two CBS domains (being limited to intramolecular
domains) is not a structural determinant for protein multimerization. A second conclusion that can be drawn from the
available structures of CBS-containing enzymes is that CBS
domains are not part of the catalytic center. In S. pyogenes
IMPDH, the CBS domains are at the periphery of the enzyme
complex and outside the catalytic center (78). Similarly, the
truncated human CBS, lacking the CBS domains, is still
catalytically active, although the regulation of the enzyme
activity is disturbed (50).
C1371
Invited Review
C1372
STRUCTURE AND FUNCTION OF CBS DOMAINS
AMP-Activated Protein Kinase
The AMPK cascade functions as a sensor of cellular energy
content, and it plays an important role in restoring the cellular
ATP balance during periods of metabolic stress (see Fig. 3B)
(8). AMPK activation occurs when the cellular energy content
drops (low ATP, high AMP) and requires a dual signal (see
Fig. 4): binding of AMP and phosphorylation by an upstream
kinase, the AMPK kinase (29). Once activated, AMPK
switches on catabolic pathways (e.g., fatty acid oxidation,
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Fig. 4. Model for the regulation of the AMP-activated protein kinase complex.
AMPK is a heterotrimer, composed of a catalytic subunit (␣) and two
regulatory subunits (␤ and ␥). The ␤-subunit functions as a scaffold on which
the ␣- and ␥-subunits assemble by binding to the conserved KIS and ASC
domains, respectively. When the CBS domains are unoccupied (low AMP
levels), the AMPK complex exists in the inactive conformation (top), in which
an autoinhibitory region in the ␣-subunit binds to the kinase domain on the
same subunit, thereby blocking activation by AMPKK and access to downstream substrates. Binding of AMP to the CBS domains activates AMPK
(bottom). The AMP-bound CBS domains interact with the autoinhibitory
region, thereby dislodging this region from the kinase domain, which, after
phosphorylation by AMPKK, becomes fully active. See also Refs. 8 and 27.
glycolysis) and switches off anabolic pathways (e.g., fatty acid
and cholesterol synthesis, glycogen synthesis) by phosphorylation of regulatory metabolic enzymes in an effort to raise the
cellular ATP level (for review, see Ref. 27). This wide range of
effects not only affects the cellular energy management but
also plays an important role in regulating whole body energy
storage and expenditure. Recently, AMPK activity has been
linked to type 2 diabetes and the metabolic syndrome, which
has led to the concept of AMPK activators as new drugs to treat
type 2 diabetes, obesity, and the metabolic syndrome (55, 77).
AMPK is a heterotrimer, composed of a catalytic subunit (␣)
and two regulatory subunits (␤ and ␥). In mammals, different
isoforms of each subunit have been identified: ␣1, ␣2, ␤1, ␤2,
␥1, ␥2, and ␥3 (8, 73, 74). The ␥-subunit contains four CBS
domains, as shown in Fig. 4. Recently it has been reported that
the two CBS pairs in the ␥-subunit provide allosteric binding
sites for AMP and ATP (68), as originally proposed by Daniel
and Carling (15). The observation that both nucleotides, ATP
as well as AMP, bind to AMPK is consistent with earlier
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assays with the isolated CBS pair of cystathionine ␤-synthase
showed that AdoMet binds to the cystathionine ␤-synthase
CBS pair with a dissociation constant of 34 ␮M. A very likely
interpretation of these data is that the COOH-terminal CBS
domains exert an autoinhibitory effect on CBS activity and that
AdoMet binding to the CBS domains induces a conformational
change, thereby relieving the autoinhibitory clamp.
In addition to their regulatory role, the CBS pairs in cystathionine ␤-synthase also affect the multimeric nature of the
enzyme. Removal of the COOH-terminal CBS domains converts the human and yeast CBS homotetramer into a homodimer (37, 39) with a higher basal activity, but resistant to
AdoMet activation (in the case of the human enzyme). Given
the currently available structural data, it is unlikely that a CBS
domain in one subunit interacts with a CBS domain in another
subunit, but an association between two CBS pairs located in
two different subunits cannot be excluded and could contribute, either directly or indirectly, to the formation of a higherorder protein complex. However, structural analysis of the
CBS complex is required to unequivocally elucidate the role of
CBS domains/pairs in the tetramerization process.
CBS deficiency due to loss-of-function mutations in the CBS
gene results in homocystinuria, a genetic disorder that is
characterized biochemically by elevated plasma levels of homocysteine and clinically by mental retardation, lens dislocation, skeletal abnormalities, and endothelial dysfunction (52).
A total of 131 different homocystinuria-causing mutations
have been identified (see http://www.uchsc.edu/cbs/cbsdata/
cbsmain.htm), some of which induce point mutation in either
of the CBS domains: e.g., I435T, D444N, and S466L (40, 45,
69). A common functional feature of the mutations in the CBS
domains is that they abolish or strongly reduce activation by
AdoMet (40). Interestingly, these mutations can be discriminated based on their effect on AdoMet binding. The D444N
mutation prevents AdoMet binding to the CBS domains (68),
whereas the I435T and S466L mutants are still capable of
AdoMet binding (35). Intriguingly, when the cystathionine
␤-synthase domains are modeled on the bacterial crystal structure, these mutations map to different locations, with respect to
the central cleft. The D444 residue maps to the beginning of
the first ␤-sheet and lines the central cleft. In contrast, I435 and
S466 reside in the ␣1- and ␣2-helix, respectively, and do not
contact the central cleft. Provided that the central cleft corresponds to the AdoMet binding site, the different positioning of
the I435, D444, and S466 residues would nicely explain their
opposing effects on AdoMet binding. At present it is not
known how CBS domain mutations that do not interfere with
AdoMet binding abolish enzyme activation. One possibility is
that these mutations disturb the AdoMet-induced conformational change that leads to disinhibition of the enzyme.
Invited Review
STRUCTURE AND FUNCTION OF CBS DOMAINS
IMPDH
IMPDH is the key enzyme in the de novo guanosine
nucleotide biosynthesis. It catalyzes the rate-limiting, NADdependent oxidation of IMP to xanthosine-5⬘-monophosphate
(XMP), which is subsequently aminated to guanosine-5⬘monophosphate, and converted to GTP or dGTP (14, 33, 72,
76). GTP and dGTP are essential nucleotides for DNA and
RNA synthesis, and numerous studies (12, 32, 33, 63, 70, 76)
have reported a relationship between increased cell proliferation and increased IMPDH activity, both in normal tissues and
in malignant cells. This is of particular importance for B and T
lymphocytes, which depend on the de novo pathway, and thus
IMPDH activity, to initiate a proliferative response after mitogen or antigen stimulation (1). Moreover, IMPDH activity may
also be linked to malignant cell transformation or tumor progression (11). Therefore, IMPDH is considered a pharmacological target for anticancer and immunosuppressive chemotherapy (e.g., mycophenolic acid) (56, 76).
Mammals contain two IMPDH isoforms (types I and II). The
human isoforms are 514 amino acids long and are 84% identical in amino acid sequence. They are differentially expressed
in normal and neoplastic human tissues (59). Gene-targeting
experiments in mice showed that type I IMPDH is unable to
substitute for type II because deletion of the type II gene leads
to embryonic death at day 9 of gestation (24). In contrast,
targeted disruption of the type I gene led to a milder and viable
phenotype (25). Early reports (57, 58) showed that type I is
constitutively expressed and is the preponderant isoform in
normal cells, whereas type II is selectively upregulated in
neoplastic and replicating cells. Moreover, in neoplastic cells,
which are induced to differentiate, the level of the type II
transcript is selectively downregulated to a level below that of
type I (57, 58). However, more recent studies (16, 34) have
indicated an increase in the mRNA levels of both isoforms in
human lymphocytes stimulated with T cell mitogens. This
means that it is not yet clear which isoform is the most
important therapeutic target.
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The native enzyme is a homotetramer, and X-ray structure of
the hamster type II IMPDH [which differs from human type II
in only six amino acids (11, 59)] revealed two domains in the
IMPDH monomer: a core domain, which forms an eightstranded ␣/␤ barrel, and a subdomain, which consists of a CBS
pair (71, 78). An identical structure in human IMPDH II was
confirmed by Colby et al. (10). The core domains of the four
subunits associate to form a central catalytic site, whereas the
four CBS pairs are located at the periphery of the enzyme
complex. Several observations indicate that the CBS pairs in
the subdomain are dispensable for IMPDH function or tetramerization in vitro. First, a deletion mutant of human
IMPDH II that lacks the CBS subdomain remains fully active
in vitro (71). Second, Nimmesgern and coworkers (60) also
showed that the tetrameric conformation is preserved in human
IMPDH II that contains only the core domains. Nevertheless,
mutations in the CBS domains of IMPDH I underlie an autosomal
dominant form of retinitis pigmentosa (RP10), which suggests a
critical in vivo function of these domains (5, 38).
Scott et al. (68) recently showed that the CBS pair of
IMPDH II binds ATP in vitro and that the tetrameric IMPDH
binds ATP in a positive, cooperative way (see Fig. 5). This
observation can be explained by assuming that binding of the
first ATP molecule to a CBS pair causes a conformational
change, which increases the affinity for ATP of the remaining
CBS pairs. They also observed that IMPDH was activated by
ATP, which has never been reported before. ATP binding and
activation was abolished by a single amino acid exchange
(R224P) in the second CBS domain of IMPDH II. Interestingly, the R224P mutation corresponds to an RP10-causing
mutation in IMPDH I (5). This provides strong evidence that
ATP binding to the CBS domains allosterically activates
IMPDH and consequently XMP synthesis (68). If so, this
mechanism would couple the GTP/dGTP biosynthesis to the
cellular energy status (high ATP levels).
CLC Chloride Channels
Chloride channels belonging to the CLC family sustain a
wide variety of cellular functions, including membrane excitability, synaptic communication, transepithelial transport, cell
volume regulation, cell proliferation, and acidification of endosomes and lysosomes (for review, see Ref. 36). The mammalian CLC family contains nine members (ClC-1–ClC-7,
ClC-Ka, and ClC-Kb), which can be subdivided into three
branches on the basis of sequence homology.
X-ray diffraction studies of bacterial CLC proteins have
revealed a dimeric two-pore structure in which each pore is
formed by a single subunit (17). In view of the sequence
identity between prokaryote and eukaryote CLC proteins and
in view of functional evidence consistent with two independent
conduction pathways in mammalian ClC-1 (65), it is very
likely that eukaryotic CLC proteins adopt a dimeric, doublebarreled structure. In contrast to bacterial CLC proteins, which
have a short COOH terminus without CBS domains (17, 48,
54), all eukaryotic CLC proteins possess a long COOH-terminal cytoplasmic region that contains two CBS domains (3, 62).
Because bacterial CLC proteins dimerize in the absence of
CBS domains (48, 54), it can be inferred that the CBS domains
are not required for dimerization. Experiments with COOHterminal truncation mutants of mammalian ClC-1 have indeed
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observations that high concentrations of ATP antagonize activation of AMPK by AMP (13).
In humans, mutations in the CBS domains of the AMPK
␥2-subunit cause a glycogen storage disease, which is clinically
expressed as a familial hypertrophic cardiomyopathy with
conduction anomalies (Wolff-Parkinson-White syndrome) (2,
4, 22, 23). In pigs, but not in humans, a mutation in the CBS
domains of the ␥3-subunit causes an abnormally high glycogen
content in skeletal muscle (51). Both disease phenotypes are
compatible with a sensor function for the CBS domains.
Because AMPK with mutant CBS domains would be insensitive to energy depletion signals (high AMP, low ATP), it
would fail to activate the compensatory metabolic pathways
(increase in energy production and decrease in energy utilization), which would explain the increased glycogen content in
cardiac (human) or skeletal (pig) muscle. However, there is
still some controversy with respect to the functional effect that
the CBS mutations exert on AMPK activity. Some claim that
the mutant AMPK is constitutively active (2, 26), but other
results suggest that the major effect of the mutations is to
reduce activation of AMPK in response to metabolic stress
(15, 68).
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Invited Review
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STRUCTURE AND FUNCTION OF CBS DOMAINS
confirmed that the CBS domains are dispensable for protein
dimerization (18). Furthermore, because the CBS domains
reside in the cytosol, they do not contribute to the core
structure of the permeation pathway, which is formed by a
subset of the membrane-embedded ␣-helices A to R. Although
the CBS domains are located outside the membrane-spanning
part of the channel, they may still modulate key properties such
as channel gating (see below). Indeed, the cytosolic COOH
terminus containing the CBS domains is an extension of the
ultimate membrane helix R, which is part of the selectivity
filter (17). This structural link allows for the direct transmission of interactions at the CBS domains to the channel pore.
Finally, it should be mentioned that the CBS1 and CBS2
domains are separated by an intervening region, which varies
in length between the different CLC isoforms. Whether this
intervening region affects the function of the CBS domains
remains to be shown.
The functional role of CBS domains in CLC channel function remains largely unresolved and even controversial. However, it is clear that CBS domains in human CLC channels are
required for CLC function and/or expression because mutations in CBS domains of ClC-1, -2, -5, and -7 and ClC-Kb
AJP-Cell Physiol • VOL
result in specific diseases caused by CLC dysfunction (9, 28,
41, 42, 46, 64). The requirement for intact CBS domains is also
corroborated by expression studies of ClC-0 or ClC-1 channels
lacking the second CBS domain. Expression of CBS2-deficient
truncation mutants in Xenopus laevis oocytes failed to generate
typical ClC-0 or ClC-1 membrane currents, whereas coexpression of the truncated proteins with the CBS2-containing
COOH-terminal fragment restored channel function (49, 66).
One set of data implicates CBS domains in the intracellular
trafficking of CLC proteins. First, when expressing mouse
ClC-5 mutants in CHO-K1 and IMCD-3 cells, Carr et al. (6)
discovered that truncation mutants with a disrupted second
CBS domain are retained in a perinuclear compartment instead
of being delivered to acidic endosomes as was the fate of
wild-type ClC-5. Second, disruption of the second CBS domain by alanine scanning led to mislocalization of the Saccharomyces cerevisiae CLC (Gef1p) (67). Wild-type Gef1p is
sorted to the Golgi apparatus, but Gef1p with a mutated CBS2
is retained in a compartment that also stains for Kar2p, a
marker for the endoplasmic reticulum. Third, it was recently
shown in expression studies in Xenopus laevis oocytes that
ClC-1 proteins truncated after the first CBS domain did not
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Fig. 5. Overview of the CBS domains. WPW, Wolff-Parkinson-White; CLC, chloride channel; and OMIM, Online Mendelian Inheritance in Man.
Invited Review
STRUCTURE AND FUNCTION OF CBS DOMAINS
AJP-Cell Physiol • VOL
sorting and gating of ClC-1, but it is presently not clear how
these discrepancies should be resolved. For the sake of clarity,
it should be mentioned that both data sets were obtained under
different experimental conditions. Hebeisen et al. transiently
overexpressed wild-type and mutant ClC-1 (either “naked” or
as fusion protein with yellow/cyan fluorescent protein) in
tsA201 cells. In contrast, Estevez et al. overexpressed wildtype and mutant ClC-1/0 with or without epitope tags in
Xenopus oocytes. However, it remains to be established to
which extent the different methodologies can explain the apparent differences between both studies. Nevertheless, a couple
of common conclusions can be deduced despite the partially
incongruent data. First, formation of a COOH-terminal CBS
pair is not required for plasma membrane delivery of ClC-1.
Indeed, either CBS1 (30) or CBS2 (18) can be in-frame deleted
without compromising surface expression of the mutant channel, indicating that the critical sorting signal for plasma membrane delivery is not located within the CBS pair. Second, the
CBS domains in ClC-1 function as interchangeable units
because CBS1 or CBS2 could be replaced by CBS domains
of other CLC channels or of other CBS-containing proteins.
This is consistent with the modular structure of a CBS pair
as revealed by X-ray diffraction of bacterial CBS proteins.
Third, CBS domains in ClC-1 modulate the functional
behavior of the channel.
Finally, an unresolved question with respect to the CBS
domains in CLC channels is whether they bind any ligands
(small molecules, proteins) and, if so, whether this has any
functional implications. On the basis of an in vitro binding
assay using bacterially expressed CBS pairs, Scott et al. (68)
proposed that ATP is the natural ligand for CLC CBS pairs.
Because the CBS domains of all CLC channels are predicted to
reside in the cytosol, Scott et al. (68) hypothesized that CLC
CBS pairs function as cellular energy/ATP sensors and that
ATP binding to the CLC CBS domains is required for channel
gating. Interestingly, mutations in the ClC-2 CBS domains,
e.g., G715E (associated with generalized epilepsy, see Ref. 28)
and G826D (equivalent to a ClC-1 mutation causing congenital
myotonia, see Ref. 64), strongly diminish the in vitro ATP
binding, suggesting a causal link between reduced ATP binding and channel dysfunction. However, it remains unclear
whether ATP binding is a universal property of all CLC CBS
domains and, if so, to which extent ATP binding modulates the
functional properties of CLC channels. Of note, there is some
circumstantial evidence in favor of this hypothesis. For example, human ClC-4 only generates a CLC when ATP or a
nonhydrolyzable analog is provided at the cytoplasmatic side
(75), but it remains to be shown that ATP mediates its effect
via binding to one of the ClC-4 CBS domains.
In conclusion, intramolecular association of two CBS domains generates a CBS pair with a conserved tertiary structure
in spite of the rather low sequence identity of individual CBS
domains. The bacterial crystal structures reveal a cleft between
the two CBS domains, which may correspond to the binding
site for the various ligands (ATP, AMP, AdoMet) that have
been reported in in vitro studies of mammalian CBS domains.
In vivo corroboration of the ligand binding properties is required, but the hypothesis that CBS domains function as
metabolic sensors by sampling the cytosolic AdoMet, ATP,
and/or AMP levels provides an interesting and unifying concept that couples domain function to the specific function of the
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reach the plasma membrane and that coexpression with the
COOH-terminal fragment containing the second CBS domains
restored plasma membrane delivery (18). Functional reconstitution and hence plasma membrane delivery of the split ClC-1
channel was observed only when the COOH-terminal fragment
contained an intact CBS2 domain (18). However, one should
be cautious in deducing from these data that CBS2 contains a
sorting signal for plasma membrane delivery. Indeed, in-frame
deletion of CBS2 did not affect the functional expression of
ClC-1 channels, indicating that CBS2 as such is dispensable
for membrane sorting and channel function (18, 31). One
interpretation of these data is that ClC-1 contains an as yet
uncharacterized sorting signal for plasma membrane delivery
or retention that does not reside in the CBS2 domain and that
functions both in the single peptide channel and in the split
channel configuration. However, in the split channel, the two
fragments must associate via a CBS1-CBS2 interaction to
bring the sorting signal into a “sorting-competent” position. In
contrast, in the single peptide channel, the sorting signal is
correctly positioned independently of the CBS interaction.
That the CBS domains in ClC-1 do not contain a plasma
membrane sorting signal sensu stricto can also be concluded
from the experiments reported by Hebeisen et al. (30). In
contrast to Estevez et al. (18), they observed that COOHterminal truncation mutants of ClC-1, when overexpressed in
tsA201 cells (a SV40-transformed variant of human embryonic
kidney HEK-293), were inserted into the plasma membrane
even if they lacked CBS domains. To conclude, although some
of the available data suggest that CBS domains influence,
either directly or indirectly, the subcellular localization of CLC
proteins, it is still too early to draw any firm conclusion about
the underlying mechanism. Finally, one should always bear in
mind that the missorting of a mutant protein does not necessarily reflect the loss of a sorting signal but could be secondary
to malfolding of the mutant protein and retention of the
malfolded protein by the quality control system in the endoplasmic reticulum.
A second set of data suggests that CBS domains may also
affect the functional properties of CLC channels. For example,
Estevez et al. (18) identified specific point mutations in the
CBS2 domain that changed the common slow gate of ClC-1
and ClC-0. In particular, it was shown that mutation of E763,
which corresponds to a conserved acidic residue in many CBS
domains, abolished the common slow gate of ClC-0 without
interfering with the fast gate of the individual protopores.
These experiments are consistent with previous observations,
which revealed that exchanging the CBS domains between, on
the one hand, ClC-0, and on the other hand, ClC-1 or ClC-2,
modified the common gate of ClC-0 (21). Consistent with a
role for CBS domains in channel function, Hebeisen et al. (30)
reported that CBS domains are required for proper functioning
of the ClC-1 channel. However, in contrast to Estevez et al.,
they concluded that CBS domains do not (or only minimally)
affect the gating behavior of ClC-1 and that the CBS domains
act only in cis, i.e., on the corresponding protopore and not on
the other protopore of the channel dimer. Finally, Hebeisen et
al. also concluded that CBS1 and CBS2 do not bind to each
other and that a single CBS domain is sufficient to support
ClC-1 channel function.
At first sight, the studies by Estevez et al. and Hebeisen et al.
offer some divergent views on the role of CBS domains in
C1375
Invited Review
C1376
STRUCTURE AND FUNCTION OF CBS DOMAINS
NOTE ADDED IN PROOF After this review was accepted, a
paper was published which shows that ATP inhibits ClC-1 by
shifting the voltage dependence and that specific mutations in
the CBS domains abolish or reduce the ATP inhibition (3a).
ACKNOWLEDGMENTS
The authors’ research is supported by the Forton Foundation (Koning
Boudewijn Stichting, Belgium), the Fund for Scientific Research (Flanders,
Belgium), and the Bijzonder Onderzoeksfonds of the K.U. Leuven.
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STRUCTURE AND FUNCTION OF CBS DOMAINS