From the Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro, Tokyo 153, Japan
Received for publication, September 1, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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A novel gene encoding an adenylyl cyclase,
designated cyaG, was identified in the filamentous
cyanobacterium Spirulina platensis. The predicted
amino acid sequence of the C-terminal region of cyaG was
similar to the catalytic domains of Class III adenylyl and guanylyl
cyclases. The N-terminal region next to the catalytic domain of CyaG
was similar to the dimerization domain, which is highly conserved among
guanylyl cyclases. As a whole, CyaG is more closely related to guanylyl
cyclases than to adenylyl cyclases in its primary structure. The
catalytic domain of CyaG was expressed in Escherichia coli
and partially purified. CyaG showed adenylyl cyclase (but not guanylyl
cyclase) activity. By site-directed mutagenesis of three amino acid
residues (Lys533, Ile603, and
Asp605) within the purine ring recognition site of CyaG to
Glu, Arg, and Cys, respectively, CyaG was transformed to a guanylyl
cyclase that produced cGMP instead of cAMP. Thus having properties of both cyclases, CyaG may therefore represent a critical position in the
evolution of Class III adenylyl and guanylyl cyclases.
Adenylyl cyclase (AC),1
which synthesizes the signaling molecule cAMP, plays an important role
in regulating various cell processes. So far, many genes encoding ACs
have been isolated from a number of organisms. ACs can be separated
into three classes according to the primary structure of the catalytic
domains (1). Class I ACs are mainly composed of enterobacterial ACs.
Class II ACs have been found in pathogenic bacteria and are activated
by the eukaryotic cofactors calmodulin (2, 3) and an as yet
unidentified factor (4). Class III ACs are widely distributed from
bacteria to mammals and form the biggest family of ACs. Recently, novel ACs showing a unique primary structure have been found in
archaebacteria. These are proposed to represent a fourth class of ACs
(5). No sequence homology can be found among all four AC classes.
cGMP also functions as an important signaling molecule and is
synthesized by guanylyl cyclase (GC). A large number of GC genes have
been isolated from eukaryotes. However, only one GC gene has been
reported in prokaryotes (6). In contrast to the divergent nature of the
catalytic domains of ACs, the catalytic domains of all GCs identified
are homologous to those of Class III ACs. Thus, Class III ACs and GCs
are thought to have evolved from a common ancestor.
Crystal structures of the catalytic domains of mammalian Class III ACs
have been solved (7, 8). Based on these structures and modeling
studies, essential residues required for substrate binding (ATP or GTP)
have been identified (9). The amino acid residues that form hydrogen
bonds with adenine and guanine are conserved among Class III ACs and
GCs (9). By exchanging these residues, GCs can be converted to ACs (10,
11). Similarly, an AC has been converted to a nonselective purine
nucleotide cyclase (11).
The N-terminal regions next to the catalytic domain (~50 amino acid
residues) are conserved among GCs, but not among Class III ACs. These
regions are predicted to form an amphipathic We have isolated six AC genes from the cyanobacterium Spirulina
platensis by functional screening using Escherichia
coli defective in its AC gene (14). We previously characterized
two AC genes, cyaA and cyaC (14, 15). The
cyaA gene encodes a putative membrane-bound AC, whereas the
cyaC gene encode an AC that is activated in response to
autophosphorylation (14, 16). In this study, we describe the
characterization of a third AC gene from S. platensis,
cyaG. The amino acid sequence of the putative catalytic
domain of CyaG was found to be homologous to those of the catalytic
domains of Class III ACs and GCs. Interestingly, the upstream regions
next to the catalytic domain of CyaG are homologous to the dimerization domain of GCs. We investigated the catalytic activity of CyaG by
generating wild-type and mutant recombinant proteins. We found that
CyaG, a strict adenylyl cyclase, was transformed to a guanylyl cyclase
by replacing three key amino acid residues within the substrate-binding
site. Based on the results, the evolution of Class III ACs and GCs is discussed.
Bacterial Strains, Plasmids, and Growth Media--
The E. coli strains JM109 (recA1, endA1, gyrA96, thi, hsdR17
(rK Genetic Methods--
Plasmid preparations, restriction enzyme
digestions, and ligations were performed as described by Maniatis
et al. (17).
Cloning of the cyaG Gene from S. platensis--
The genomic
library of S. platensis was screened by functional
complementation of the mutant of E. coli MK1010 defective in AC activity. The positive clone pCYA68 was isolated as described in
our previous report (14).
To isolate overlapping fragments, the genomic library of S. platensis was produced with a phage vector (15). Recombinant phages were screened for the presence of the desired insert using the
BlnI-BamHI fragment (~0.5 kilobase pair)
of pCYA68 as a probe. Following secondary screening, positive
recombinant phages were converted to pBK-CMV plasmids by in
vivo excision using the Stratagene protocol.
DNA Sequencing and Analysis--
A 2.6-kilobase pair DNA
fragment containing the 2.0-kilobase pair DNA insert of S. platensis was excised from pCYA68 with HindIII and
SalI digestions and subcloned into the
HindIII-SalI site of pBluescript II
KS+ (Stratagene). A 2.0-kilobase pair fragment was
excised from pBKG1 with BlnI and DraI, treated
with the Klenow fragment to fill the ends, and subcloned into the
EcoRV site of pBluescript SK+ (Stratagene).
Deletions were constructed using exonuclease III and mung bean
nuclease. The nucleotide sequence was determined by the dideoxy chain
termination method using a DNA sequencer (Model 373A) and a
Taq dideoxy terminator cycle sequencing kit (both from
Applied Biosystems). The DNA sequence and predicted amino acid sequence
were analyzed with DNASTAR software. Data base searches for similarity
to other proteins were performed using the GenBankTM BLAST server.
Construction of the Expression Plasmid for the GST-CyaG-CD
Protein--
A BamHI restriction site was introduced
immediately upstream of the G1249 base (the second
letter of the codon encoding Ser380), and an
EcoRI restriction site was introduced 75 base pairs downstream of the stop codon using PCR. The PCR product was ligated into the pGEX-3X vector digested with BamHI and
EcoRI. The resulting plasmid, pGEX-CyaG-CD (where CD is
catalytic and dimerization domains), contained a portion of the
cyaG gene fused to the GST tag sequence from pGEX-3X, which
is placed under the control of the tac promoter. The portion
of pGEX-CyaG-CD derived from the PCR fragment was sequenced. We found
that the A1610 base was altered to C by PCR error. However,
this replacement was fortunately a silent mutation, which did not
result in the replacement of this amino acid residue.
Expression and Purification of GST-CyaG-CD--
The
transformants (TP2339 cells harboring pGEX-CyaG-CD) were grown at
27 °C in LB medium (1.5 liter) supplemented with ampicillin (100 µg/ml). The recombinant gene was expressed by inducing cells (A600 = 0.8) with 0.1 mM
isopropyl- Site-directed Mutagenesis--
The GST-CyaG-CD-K533E mutant was
constructed with mismatch oligonucleotides and PCR using ExTaq DNA
polymerase (Takara). PCRs were performed with primers P1
(5'-AAGAAGTCACTATTTTATTTG-3') and P3 (5'-CATAAACTCGAGAAGATTGAAAC-3') or
primers P2 (5'-GTTTCAATCTTCTCGAGTTTATG-3') and P4
(5'-GATCGTCAGTCAGTCAC-3'). The resulting PCR products were mixed, and
PCR was repeated with primers P1 and P4. The PCR product was digested
with EcoRI and BamHI (partial) and ligated into
pGEX-CyaG-CD digested with EcoRI and BamHI
(partial). The resulting construct, pGEX-CyaG-CD-E, contained the
cyaG gene with the CyaG K533E mutation.
The GST-CyaG-CD-K533E/I603R/D605C mutant was constructed with mismatch
oligonucleotides and PCR using ExTaq DNA polymerase. PCRs were
performed with primers P1 and P5 (5'-CCCCATAAACAGTATCTGAATTTTTTAAT-3') or primers P4 and P6 (5'-ATTAAAAAATTCAGATACTGTTTATGGGG-3'). The PCR
product was ligated into the pCRII vector (Invitrogen) to make pCRERC.
The EcoRI-XhoI fragment of pCRERC (500 base
pairs) was ligated into pGEX-CyaG-CD-E digested with EcoRI
and XhoI. The resulting construct, pGEX-CyaG-CD-ERC,
contained the cyaG gene with the CyaG K533E, I603R, and
D605C mutations. The portions of pGEX-CyaG-CD-E and pGEX-CyaG-CD-ERC
derived from the PCR fragments were sequenced to ensure that no other
mutations had arisen.
AC and GC Activity Assays--
Unless otherwise stated, the
in vitro AC or GC reaction was performed in 0.1 ml of 50 mM Tris-HCl (pH 7.5) containing 1 mM MnCl2, 1 mM dithiothreitol, 0.1% (w/v) bovine
serum albumin, and 1 mM ATP or GTP, respectively. The
reactions were performed at 37 °C for 20 min and terminated by
addition of 1 ml of 5% (w/v) trichloroacetic acid. After
trichloroacetic acid was removed from each reaction mixture by
extraction with ethyl ether, samples were lyophilized. cAMP or cGMP
content was measured by an enzyme immunoassay system (EIA system,
Amersham Pharmacia Biotech) according to the manufacturer's protocol.
Other Analytical Methods--
Protein content was measured by
the method of Bradford (18) as described in the instructions of the
Bio-Rad protein assay kit. Bovine serum albumin was used as the
standard. Gel electrophoresis on polyacrylamide gel containing 0.1%
SDS was carried out following the method of Laemmli (19).
Isolation of the S. platensis AC Gene--
The genomic library of
S. platensis was screened by functional complementation of
the E. coli strain MK1010, which is defective in AC activity
and therefore lacks cAMP. Seven distinct positive clones were isolated
(14). A significant level of cAMP was detected in cells transformed
with pCYA68, one of the positive clones, suggesting that pCYA68
contains an AC gene of S. platensis (14). We determined the
nucleotide sequence of the insert region of pCYA68. An ORF was found in
the sequence, but the N terminus of the ORF was absent in pCYA68. To
isolate an overlapping clone, we screened an S. platensis
genomic library as described under "Experimental Procedures." A
positive clone (pBKG1) was isolated, and restriction mapping confirmed
that this clone contained the N terminus of the ORF (data not shown).
DNA Sequencing--
A nucleotide sequence (2449 base pairs) was
determined by sequencing the insert region of pCYA68 and a
DraI-BlnI fragment of pBKG1 (Fig.
1). An ORF that encodes a polypeptide of
671 amino acids with a predicted molecular mass of 75,365 Da was
identified in this sequence. The C-terminal region of the predicted
amino acid sequence was homologous to the catalytic domains of Class III ACs and GCs. The ORF was therefore named cyaG. A
hydropathy profile shows that CyaG contains two hydrophobic regions
(Fig. 1, underlined). One
(Met1-Gly31) is near the N-terminal end, and
the other (Ile351-Ile376) is located in the
central region of the protein. The N-terminal hydrophobic region is
closely related to the signal peptides of bacteria required for the
targeting of proteins to membranes (20-22). The latter hydrophobic
region probably represents a membrane-spanning region.
Comparison of the C-terminal Region of CyaG with the Catalytic
Domains of Class III ACs and GCs--
The amino acid sequence of CyaG
exhibited similarities to the catalytic domains of Class III ACs and
GCs. Fig. 2A shows the alignment of the C-terminal region
(Val487-Gly663) of CyaG with the catalytic
domains of various Class III ACs and GCs. The putative catalytic domain
of CyaG has 29-44% identity to those of Class III ACs and 35-48%
identity to those of GCs. Asterisks in Fig. 2A
indicate essential amino acid residues required for interacting with
the adenine or guanine substrate ring (9). The essential amino acid
residues present in CyaG were more similar to those of Class III ACs
than to those of GCs.
The complete genome sequence of a filamentous cyanobacterium,
Anabaena sp. PCC 7120, has recently been determined.
In addition to those five AC genes that have already been isolated from
Anabaena 7120 (23), we found a novel AC gene by searching
the database at the Kazusa DNA Institute and named this gene
cyaE. Among the six Anabaena ACs, the catalytic
domain of CyaG is more similar to that of the novel Anabaena
AC, CyaE (Fig. 2A).
We performed a phylogenetic analysis of the catalytic domains of the
Class III ACs and GCs. The catalytic domain of CyaG was found to be
more closely related to those of eukaryotic Class III ACs and GCs than
to those of cyanobacterial and bacterial ACs (Fig. 2B).
Existence of the Dimerization Domain in CyaG and Anabaena CyaE,
Commonly Found in GCs--
We detected a unique amino acid sequence
homologous to the so-called dimerization domain of GCs in the
N-terminal region next to the catalytic domains of CyaG and
Anabaena CyaE (Fig. 3). The dimerization domain is present in all GCs except two exceptional GCs (6, 24), but not in Class III ACs. Rat GC-A forms a homodimer by
interacting with the dimerization domain of each subunit (12). The
domain is predicted to form an amphipathic
Fig. 4 shows domain organizations of
CyaG, Spirulina CyaC, rat AC2, rat soluble GC (GC-S)
Expression and Purification of CyaG--
To investigate whether
CyaG exhibits AC and/or GC activity, we purified the C-terminal domain
of CyaG, which contains both the catalytic and dimerization domains, as
a GST fusion protein (GST-CyaG-CD). We failed to construct the
expression vector encoding the GST-CyaG-CD protein (pGEX-CyaG-CD) when
using an E. coli strain, JM109
(cya+, crp+), for
cloning. This was probably due to an inhibitory effect of the high
concentration of cAMP on the growth of E. coli (27). By
using strain TP2339 (cya-,
crp
The transformants (TP2339 cells harboring pGEX-CyaG-CD) were grown at
27 °C and induced for the production of the GST-CyaG-CD protein by
the addition of 0.1 mM
isopropyl- AC and GC Activities of the GST-CyaG-CD Protein--
The effects
of divalent cations on the AC activity of the GST-CyaG-CD protein were
measured. The specific activities, Mg2+- and
Mn2+-dependent activities (1 mM
MgCl2 and 1 mM MnCl2), were 0.036 and 11 nmol of cAMP formed per min/mg in the presence of 10 µM ATP, respectively. The observation that
Mn2+ stimulates CyaG catalytic activity is similar to
results found for Class III ACs and GCs from S. platensis
and eukaryotes (15, 28, 29). AC and GC activities were assayed in the
presence of Mn2+ as described under "Experimental
Procedures." Significant AC activity was detected, but no GC
activity was detected (Fig. 6).
Site-directed Mutations within the Predicted Nucleotide-binding
Site of CyaG--
The cyclase catalytic consensus sequence is
conserved among the Class III AC and GC families (30). Residues
interacting with the ribose and triphosphate of ATP or GTP and the
metal ion are conserved between Class III ACs and GCs. However,
interactions with the purine ring are different between the ATP- and
GTP-binding sites of Class III ACs and GCs (9). We investigated whether the substrate of CyaG could be changed from ATP to GTP by site-directed mutagenesis of the purine ring-binding site. Liu et al. (9) identified amino acid residues involved in recognizing the purine ring
of the substrate by a modeling study based on the three-dimensional structure of a mammalian AC (8). Lys533,
Ile603, and Asp605 of CyaG correspond to the
amino acid residues related to purine ring binding. A mutant CyaG
protein (GST-CyaG-CD-K533E) in which Lys533 was replaced by
glutamate was produced as described under "Experimental Procedures." Lys533 is replaced by glutamate in all GCs
(1). We found that GST-CyaG-CD-K533E lost AC activity completely, and
no GC activity was detected (data not shown). Next, we changed two more
amino acid residues of GST-CyaG-CD-K533E, Ile603 and
Asp605, to arginine and cysteine, respectively. The
resultant mutant protein, GST-CyaG-CD-K533E/I603R/D605C, has triple
mutations. GST-CyaG-CD-K533E/I603R/D605C lost AC activity, but obtained
significant GC activity (Fig. 6). CyaG as an AC was therefore
changed to GC by the site-directed mutations.
In this study, we isolated a novel AC gene, cyaG, from
the cyanobacterium S. platensis. The CyaG protein contains a
catalytic domain homologous to the Class III ACs and GCs at the
C-terminal region. It contains a dimerization domain conserved among
GCs at the N-terminal side of the catalytic domain and two hydrophobic regions, one at the N terminus and the other in the middle of the
protein. The N-terminal hydrophobic region has characteristics of a
bacterial signal sequence for localizing the protein to the membrane
and would be digested after targeting. CyaG is thought to be a single
transmembrane protein.
When we performed the BLAST search to find homologies using the amino
acid sequence of the cyaG gene product, we found that the
protein sequences most closely related to the C-terminal regions of
CyaG were those of GCs rather than ACs. Since we previously showed that the pCYA68 clone containing a sequence of part of cyaG produced cAMP (14), we speculated that CyaG may exhibit GC activity as well as AC activity. Furthermore, investigating the CyaG
sequence carefully, we found the dimerization domain highly conserved
among all GCs.
Wilson and Chinkers (12) predicted that the dimerization domain formed
an amphipathic To investigate the catalytic activity of CyaG, we produced a
recombinant protein (GST-CyaG-CD) containing the catalytic and dimerization domains of CyaG. GST-CyaG-CD showed
Mn2+-stimulated AC activity, but not GC activity. We then
investigated whether CyaG could obtain GC activity after exchanging
certain amino acid residues. First, we changed Lys533 to
Glu since the Lys residue is highly conserved in ACs, but the same
position contains Glu in GCs (1). GST-CyaG-CD-K533E showed no AC
activity. GC activity was also not detectable. We therefore concluded
that additional replacements were required to obtain GC activity. In
addition to the K533E substitution, we replaced two other amino acid
residues, Ile603 and Glu605, with Arg and Cys,
respectively. Ile603 and Glu605 are conserved
within the purine ring-binding site. The triple mutant
GST-CyaG-CD-K533E/I603R/D605C was found to exhibit significant GC
activity (Fig. 6), but no AC activity. Therefore, CyaG, an AC,
was converted to a GC by replacing these three amino acid residues.
The primary structure of the catalytic domain of CyaG is more closely
related to those of eukaryotic transmembrane ACs and GCs than to those
of other cyanobacterial ACs (Fig. 2B). The catalytic domains
of cyanobacterial ACs are divided into two groups. The first group
includes CyaG and Anabaena CyaE (group I), and the second
includes the remaining cyanobacterial Cya proteins (group II). These
two groups might have evolved independently just like mammalian soluble
AC and transmembrane AC (31). Interestingly, mammalian transmembrane
ACs and a soluble AC are related to the cyanobacterial group I and II
ACs, respectively (Fig. 2B). Thus, CyaG-type ACs may
possibly share a common ancestor with the transmembrane ACs and GCs.
Class III ACs and GCs are thought to be evolutionarily related to each
other because of the similarity in the primary structures of their
catalytic domains. Two GCs that have the unusual property of lacking
the dimerization domain were recently found in Paramecium (24) and Synechocystis (6). The catalytic domain of
Paramecium GC has a topology identical to that mammalian
membrane-bound ACs (Fig. 4, rat AC2) and thus was proposed to be
evolved from a eukaryotic transmembrane AC. Synechocystis GC
(Fig. 2B, Cya2) is the first example of a prokaryotic GC
whose catalytic domain is more similar to those of bacterial ACs than
to those of eukaryotic GCs. Thus, it is likely that
Synechocystis GC has evolved from a bacterial AC. The other
GC members that contain the dimerization domain seem to have an
ancestor other than Paramecium GC and
Synechocystis GC. CyaG is a unique AC in that it contains
the dimerization domain essential for GC activity. Also, the catalytic
activity of CyaG is changed to a GC by replacing only three
amino acid residues. Based on these findings, it seems likely that most
eukaryotic GCs, with the exception of Paramecium GC, have
evolved from an AC such as CyaG that contains the dimerization domain.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix and to function
as a dimerization domain (12). Besides the importance for dimerization,
this domain has also been shown to play an important role in
stimulating human retinal GC-1 catalytic activity by GC-activating proteins (13).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, mK+), supE44, relA1,
(lac-proAB)/F' (traD36, proAB, lacIq,
lacZ
M15)) and TP2339 (aroB+,
crp
39, ilv
, cya
) were
used for cloning and expression of recombinant proteins, respectively.
Bacteria were grown in LB medium. When required, ampicillin, kanamycin,
or chloramphenicol was added at 100, 25, or 30 µg/ml, respectively.
-D-thiogalactopyranoside. The cells were
harvested after 24 h by centrifugation, washed with 50 mM Tris-HCl (pH 7.5) buffer containing 150 mM
NaCl, resuspended in 80 ml of TEG buffer (50 mM Tris-HCl
(pH 8.0), 10 mM EDTA, 10% glycerol, 0.5 M
NaCl, and 1 mM phenylmethylsulfonyl fluoride) supplemented
with 80 mg of lysozyme, and disrupted by sonication. The cell extract
was centrifuged at 16,000 × g for 10 min, and the
supernatant was further centrifuged at 150,000 × g for
40 min. The 150,000 × g supernatant was loaded onto a
glutathione-Sepharose 4B column (Amersham Pharmacia Biotech)
equilibrated with TEG buffer. The column was washed with TEG buffer,
and then the protein was eluted with 10 mM glutathione in
TEG buffer.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and deduced amino acid sequences
of the cyaG gene. Two hydrophobic regions are
underlined. The catalytic domain of AC is shown in
outlined letters. The putative dimerization domain is
double-underlined.
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Fig. 2.
Amino acid alignment and phylogenetic
analysis of the catalytic domain of CyaG. A, alignment
of CyaG with Anabaena PCC 7120 CyaE, S. platensis
CyaC, Rhizobium meliloti Cya1, rat AC2, rat atrial
natriuretic peptide receptor GC-A, human RetGC-1, rat GC-S
1 (GC-S alpha-1), and rat GC-S
1 (GC-S beta-1). The rat AC2 sequence
represents two conserved regions (C1 and C2).
Amino acid residues identical in half or more of the sequences are
shown in black, and conservative substitutions are shown in
gray. Gaps introduced for good alignment are indicated by
dashes. Numbers are amino acid positions for each
protein sequence. Amino acid residues involved in recognizing the
purine ring of ATP or GTP are indicated by asterisks.
B, phylogenetic analysis of the catalytic domains of Class
III ACs and GCs. Phylogenetic analysis was performed using the MegAlign
module of the Lasergene software package (DNASTAR, Inc.). The
DDBJ/GenBankTM/EBI Data Bank accession numbers of
the protein sequences used are as follows: human RetGC-1, M92432; human
RetGC-2, L37378; Oryzias GC, AB000899; human GC-C,
U20230; rat GC-C, M55636; rat KSGC (kinase-like
domain-containing soluble GC), U33847; human
GC-A, AAF01340; rat GC-A, J05677; Drosophila GC, L35598; sea
urchin GC, M22444; rat GC-S
1, U60835; rat GC-S
1, P20595; Drosophila AC (rutabaga),
M81887; rat AC2, M80550; rat AC5, M96159; Dictyostelium ACA,
L05496; Dictyostelium ACG, M87278; Spirulina
CyaG, D49531; Spirulina CyaC, D49692;
Anabaena CyaC, D89625; Anabaena CyaD, D89626;
Synechocystis Cya2, S74929; Anabaena CyaA,
D89622; Stigmatella CyaB, AJ223795; Spirulina
CyaA, D49530; Synechocystis Cya1, S75018;
Anabaena CyaB1, D89623; Anabaena CyaB2, D89624;
Stigmatella CyaA, AJ223796; Rhizobium Cya1,
M35096; rat soluble AC, AAD04035; Neurospora AC, Q01631; and
Saccharomyces AC, P08678.
-helix, a feature that is
associated with mediating protein-protein interactions. A helical
diagram of the putative dimerization domain of CyaG using DNASTAR
software showed that this region could form an amphipathic
-helix
(data not shown). Hydrophobic amino acid residues that form a cluster
at one side of the helix are shown in Fig. 3 (asterisks). Three mutations are identified in conserved amino acid residues in the
dimerization domain of human retinal GC-1, RetGC-1, in patients with
cone-rod dystrophy (Fig. 3, arrowheads) (25, 26). By
characterizing a mutant RetGC-1 protein with Arg substituted for Cys in
the conserved amino acids, Tucker et al. (13) showed that
mutant RetGC-1 had altered responses to GC-activating proteins and
Ca2+. They concluded that the mutation in the dimerization
domain stabilized the active form of RetGC-1. The corresponding amino acid residues were also found to be conserved in the dimerization domain of CyaG.
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Fig. 3.
Alignment of CyaG with dimerization domains
of GCs. The deduced CyaG amino acid sequence is aligned with the
dimerization domains of Anabaena CyaE, rat atrial
natriuretic peptide receptor GC-A, rat heat-stable enterotoxin receptor
GC-C, human RetGC-1 and RetGC-2, rat KSGC (kinase-like
domain-containing soluble GC), rat GC-S
1 (GC-S alpha1) and
1
(GC-S beta1), Drosophila GC, Oryzias
GC, and purple sea urchin GC. Amino acid residues identical in half or
more of the sequences are shown in black, and conservative
substitutions are shown in gray. Gaps introduced for good
alignment are indicated by dashes. Numbers are
amino acid positions for each protein sequence. Amino acid residues
important for the regulation of human RetGC-1 and for forming a
hydrophobic cluster are indicated by arrowheads and
asterisks, respectively (12, 13).
1, and rat GC-A. It is worth noting that CyaG contains a
dimerization domain like those of GCs.
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Fig. 4.
Domain organizations of CyaG, Class III ACs,
and GCs. Shown are Spirulina CyaG, Spirulina
CyaC, rat transmembrane AC2, rat GC-S 1 (GC-S
alpha-1), and rat atrial natriuretic peptide receptor GC-A. The
catalytic domains of ACs and GCs are shown as black boxes.
Dimerization domains are shown as checkered boxes.
Membrane-spanning domains are shown as gray boxes. A signal
sequence is shown as a hatched box.
), which is defective in AC and the cAMP receptor
protein, we were able to construct pGEX-CyaG-CD. This method may be
useful for expressing ACs in E. coli cells.
-D-thiogalactopyranoside. The majority of the
GST-CyaG-CD protein formed was found to be in the insoluble fraction
(data not shown). Nevertheless, the 150,000 × g
fraction, in which we detected the GST-CyaG-CD protein by
immunoblotting against GST (data not shown), was loaded onto a
glutathione-Sepharose column, and the GST-CyaG-CD protein was eluted
with 10 mM glutathione (Fig.
5, lane 4). A main band with an apparent molecular mass of 54 kDa was recognized. The size was
consistent with the predicted theoretical value for the GST fusion
protein.
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Fig. 5.
Purification of the GST-CyaG-CD protein.
SDS-polyacrylamide gel electrophoresis was carried out using a 12%
polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue
R-250. Lane 1, cell extract (minus
isopropyl- -D-thiogalactopyranoside); lane 2,
cell extract induced by addition of 0.1 mM
isopropyl-
-D-thiogalactopyranoside; lane 3,
150,000 × g supernatant; lane 4,
glutathione-Sepharose 4B column chromatography. The
arrowhead indicates the position of GST-CyaG-CD. Molecular
size standards are indicated on the left.
View larger version (34K):
[in a new window]
Fig. 6.
AC and GC activities of wild-type and mutant
CyaG proteins. AC and GC activities were measured as
described under "Experimental Procedures." Partially purified
wild-type (GST-CyaG-CD) and mutant (GST-CyaG-CD-K533E/I603R/D605C)
proteins were used for the assays. AC and GC activities are shown as
black and gray bars.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix and showed that GC activity was lost upon the
deletion of the dimerization domain. The dimerization domains of both
soluble and membrane-bound GCs are located on the N-terminal side next
to the catalytic domain. To our knowledge, the dimerization domain
exists in all GCs except two GCs recently found (6, 24), but not in
ACs. Tucker et al. (13) have shown that point mutations
within the dimerization domain of human RetGC-1 result in reduced
catalytic activity of RetGC-1 and reduced stimulation by GC-activating
protein-2. These findings indicate that the dimerization domain is
essential for the catalytic activity of GCs. CyaG contains the
dimerization domain (Fig. 3), but cannot form cGMP; and thus, CyaG
represents a unique AC with a characteristic structure of GCs.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. John Christie (Carnegie Institution of Washington) for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a research fellowship from the Japan Society for the Promotion of Science for Young Scientists (to M. K.) and by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences of Japan (to M. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number D49531.
To whom correspondence should be addressed. Tel.: 81-3-5454-6631;
Fax: 81-3-5454-4333; E-mail: cohmori@mail.ecc.u-tokyo.ac.jp.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M008006200
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ABBREVIATIONS |
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The abbreviations used are: AC, adenylyl cyclase; GC, guanylyl cyclase; GST, glutathione S-transferase; PCR, polymerase chain reaction; ORF, open reading frame.
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REFERENCES |
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