(Received for publication, March 10, 1995; and in revised form, May 5, 1995)
From the
Mutagenic analysis of the substrate binding and catalytic
domains of double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was
carried out. This nuclear enzyme is likely to be involved in the RNA
editing of glutamate-gated ion channels that are essential for fast
excitatory neurotransmission in mammalian brain. The deletion of the
first or the third of the three dsRNA binding motifs within the
substrate binding domain dramatically decreases enzyme activity,
whereas the second motif seems to be dispensable. The results indicate
that the three motifs are not functionally equivalent in the catalytic
action of DRADA. Mutation of the putative zinc-coordinating residues,
His
Double-stranded RNA (dsRNA)
Although the precise biological function(s) for this newly
discovered enzyme are currently not known, one possible role DRADA may
play is to participate in an RNA editing
process(7, 8) . Inosine is treated as G by reverse
transcriptase and also by the translation machinery(9) , and,
therefore, the A to I base editing introduced by DRADA would be
detected as A to G alterations by cDNA
sequencing(7, 8) . So far, eight examples of RNA
editing that result in alteration of an adenosine residue to guanosine
detected by cDNA sequencing have been reported (10, 11, 12) . All eight cases occur in
transcripts of certain glutamate-activated ion channel subunits in
mammalian brains. These channels respond to L-glutamate, a
major neurotransmitter, and mediate fast excitatory synaptic
responses(13) . All of the amino acid changes introduced by the
RNA editing have a large physiological impact on ion conductance,
calcium permeability, and the kinetics of desensitization of the edited
glutamate receptor (GluR)
channels(10, 11, 14, 15, 16) .
In one example, an arginine residue (CGG or CIG), located in the second
transmembrane region of both
We have recently
cloned and characterized the human DRADA cDNA(21) . The deduced
primary structure of the human DRADA revealed a bipartite nuclear
localization signal(22) , three repeats of a dsRNA binding
motif
(DRBM)(23, 24, 25, 26, 27, 28, 29) ,
and the presence of sequences conserved in the catalytic center of
other deaminases(30, 31, 32) . In this study,
we have carried out deletion and site-directed mutagenesis of the dsRNA
binding domains, and also other amino acid residues that we postulate
to be involved in zinc coordination and formation of the catalytic
center of DRADA. Our results suggest that the three repeats of the
DRBMs are not functionally equivalent to each other, though their amino
acid sequences are well conserved. The first and third DRBM seem more
important for the DRADA activity, while the second DRBM can be removed
without affecting the enzymatic activity. Since the deletion of any one
DRBM did not significantly change the K
DRBM deletion mutants and also
point mutations of the catalytic domain were constructed by using the
commercially available Transformer Site-Directed Mutagenesis Kit
(Clontech, Palo Alto, CA) with some modifications. The system requires
a restriction site unique for the plasmid to be used for designing of a
selection oligo. The StuI site located 75 bp upstream of the
stop codon was chosen as a selection site. The selection oligo, StuIm, carries a single base substitution that will eliminate
the StuI site without changing any amino acid residue. Both
selection oligo and mutagenic oligo were phosphorylated using T4 DNA
polynucleotide kinase (New England Biolabs Inc.). The plasmid
pVL-DRADA140 (0.1 µg) denaturated by 0.2 N NaOH was mixed
with 0.1 µg of selection oligo StuIm and 0.1 µg of a
mutagenic oligo in a total volume of 20 µl annealing buffer
(Clontech), heated at 100 °C for 3 min, and cooled on ice. The
mutant strand was synthesized by incubating the reaction mixture with
T4 DNA polymerase (Clontech) and T4 DNA ligase (Clontech) at 37 °C
for 2 h. The wild-type plasmid DNA was linearized by StuI,
while the hybrid of wild-type strand and newly synthesized mutant
strand remained uncut. The restriction digestion products were
transformed into Escherichia coli BMH 71-18 mutS strain (Clontech). The transformants were suspended in
500 µl of Luria Broth (LB) medium (Bio101), and 20 µl of the
cell suspension were inoculated into 1.5 ml of LB containing ampicillin
100 µg/ml, and cultured at 37 °C for 6 h. The plasmid pool was
isolated by a quick alkaline method (33) and digested with StuI, once again to linearize the wild-type plasmid. The
digestion product was then transformed into E. coli DH5
All
single DRBM (amino acids 502-798) deletions,
pVL-F-DRADA140 carrying an additional FLAG epitope-tag (35) at the NH
Figure 1:
Structure of DRADA and mutants. The
structures of the wild-type (WT) human DRADA and all deletion
and point mutants used in this study are schematically shown. The
locations of the bipartite nuclear localization signal, Nuc (filled
box), three repeats of DRBM1, 2, and 3 (hatched boxes),
and the deaminase catalytic domain (striped box) are
indicated. The location of a unique StuI restriction site used
for designing the selection oligo as well as AflII and SstI sites also utilized for preparation of mutant constructs
are indicated. The lines depict the DRBM (
Figure 2:
Characterization of the C-terminal
deletion mutants. A, fluorography of the SDS-PAGE analysis for
Sf9 cells infected with the C-terminal deletion mutants is shown.
Forty-six h after infection with recombinant baculoviruses, Sf9 cells
were labeled with [
Figure 3:
Characterization of the deaminase domain
mutants. A, fluorography of the SDS-PAGE analysis; B,
analysis of the DRADA activity. The percentage of adenosine
5`-monophosphate (pA) to 5`-monophosphate (pI)
conversion by the wild-type DRADA was treated as 100; the relative
activity of the mutants is shown at the bottom. The assay
conditions were as described in Fig. 2.
Figure 4:
Characterization of the DRBM deletion
mutants. A, fluorography of the SDS-PAGE analysis; B,
analysis of the DRADA activity. The assay conditions were as described
in Fig. 2.
Purified recombinant
DRADA proteins were used for the binding assay instead of crude cell
extracts (Fig. 5). New baculovirus expression plasmids carrying
an epitope-tag peptide FLAG (35) at the NH
Figure 5:
Binding of DRADA mutants to dsRNA. Binding
of purified recombinant DRADA mutants as well as wild-type protein to
[
Three other amino
acid residues, potentially able to participate in zinc chelation, were
also mutated in this study. Of these, the mutation of Cys
Although an increasing number
of proteins that interact with RNA have been discovered in recent
years(48) , the exact nature of interaction between these
proteins and RNA is poorly understood. However, sequence-specific
interaction of DNA or RNA with protein often accompanies conformational
change of the nucleic acid so that specific bases in the nucleic acids
are in close contact with the amino acid residues responsible for the
interaction(49, 50) . Such conformational changes upon
protein binding include melting of base pairings in the double-stranded
region(51) . DRBMs not only act as the substrate binding domain
but also may generate critical conformation distortion in the dsRNA,
including disruption of the limited base pairings around the
modification site, to allow the adenosines to be exposed and accessible
to the catalytic domain of DRADA as we postulated previously (21) .
Because of the very similar amino acid sequence of
three DRBMs (Fig. 6), we originally speculated that these three
motifs together contribute evenly to the unusually high affinity of the
enzyme binding to its substrate(21) . However, our results
presented in this study suggest that the three motifs are not
equivalent and that DRBM1 and DRBM3 may play more important roles for
the enzyme action than DRBM2. In fact, the DRBM2 is dispensable at
least for the A to I conversion activity of DRADA. One possible
explanation for this unexpected result is that DRBM2 may provide a
spacer or hinge in the wild-type enzyme to give structural flexibility
for the other two DRBMs located on either sides of this motif, DRBM1
and DRBM3. The space between DRBM1 and DRBM2 or DRBM2 and DRBM3 are
almost identical, 40 and 41 amino acids, respectively, which may not be
long enough for two neighboring motifs to interact with each other upon
binding to a substrate dsRNA. Alternatively, the spacer may be
necessary to position the
Figure 6:
Difference among the three repeats of the
DRBM in DRADA. The amino acid sequences of DRADA DRBMs are shown
together with the consensus sequence obtained from alignment of 23
different repeats found in 11 DRBM-containing
proteins(21, 29) . The C-terminal region of the DRBM,
predicted to form an
One enigma
concerning the DRADA reaction is the fact that the amino group of
adenosine to be removed by DRADA through hydrolytic deamination is
engaged in Watson-Crick base pairing with uracil, forming the RNA
duplex structure of the substrate RNA. The A-U base pairing must be
disrupted prior to initiation of the deamination reaction(21) .
It may be that the specific amino acid sequence unique to DRBM1 and
DRBM3 allows them, possibly acting in concert, to destabilize or unwind
A-U base pairs, creating a local ssRNA region and non-base-paired
adenosine residues and providing easy access for the deaminase
catalytic domain of DRADA. The hypotheses discussed will be tested by
creating more mutant constructs in our future studies.
We thank Dawn H. Marchadier in the Wistar Protein
Expression Core Facility for her excellent technical assistance with
recombinant baculovirus expression, and Drs. Roger M. Burnett and John
M. Murray for helpful discussion and critical reading of this
manuscript. We also thank the Wistar Editorial Services Department for
preparing the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, Cys
, and Cys
, abolished
the DRADA activity. Similarly, the Glu
residue, predicted
to be involved in the proton transfer functions of the enzyme, was
found to be indispensable. Our results reinforce the previous proposal
that the hydrolytic deamination mechanism of DRADA may be more similar
to that of the cytidine deaminases than of adenosine deaminases.
(
)adenosine
deaminase (DRADA) deaminates adenosine residues to inosines in
dsRNAs(1, 2) . This nuclear enzyme (3) occurs
throughout the animal kingdom(3, 4) . A relatively
long double helical structure of either intermolecular or
intramolecular duplex RNA is required for efficient modification, but
the absolute minimum length of the double-stranded region for substrate
recognition may be as short as 15 bp(5) . The mechanism of A to
I conversion has been shown to be hydrolytic deamination(6) .
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) GluR-B
subunit and also a subpopulation of kainate receptor GluR-5 and -6
subunits, were generated through an RNA editing process. In this case,
an RNA editing event specifically changes the geneencoded glutamine
codon (CAG) by modification of adenosine to guanosine(12) . The
formation of a short dsRNA structure between the exonic sequence around
the editing site and the downstream intron sequence, as well as dsRNA
formed between inverted repeat structures in the intron, are essential
for RNA editing of these GluR-B transcripts(17, 18) .
Furthermore, additional adenosines are converted to guanosines in the
intron, and at the third base of another glutamine codon within the
dsRNA structure of GluR-B transcripts(17, 18) . These
two features, a requirement for dsRNA formation and multiple adenosine
modifications are the hallmark of DRADA. Finally, the presence of an
inosine residue in the edited GluR-B RNA at the Q/R site was directly
demonstrated recently, confirming that the A to G conversion detected
by cDNA sequencing is indeed derived from the A to I change of the
RNA(19, 20) . The accumulated evidence strongly
suggests that DRADA is involved in this particular RNA editing process
in mammalian brain (17, 18) .
of mutant DRADA proteins for substrate dsRNAs, DRBM1 and
DRBM3 may participate in the catalytic mechanism in addition to RNA
binding. We identified four amino acid residues essential for the
enzyme's catalytic activity. The residues His
and
Cys
, as previously predicted(22) , as well as an
additional cysteine residue, Cys
, may be involved in
zinc chelation. The residue Glu
is also found to be
indispensable, confirming its possible role in the proton transfer
reactions that occur during catalysis by DRADA.
Materials
P nuclease was purchased
from Pharmacia (Piscataway, NJ). Protein-gold protein quantitation
solution was purchased from Integrated Separation Systems (Hyde Park,
MA). FLAG epitope-tag peptide and anti-FLAG M2 affinity gel were
purchased from Eastman Kodak Co. Proteinase inhibitors
phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, and aprotinin
were obtained from Sigma.
Oligonucleotides
All oligonucleotides used for
deletion and site-directed mutagenesis are as follows:
OLStuIm, 5`-AACTACTTCAAAAAGGGCCTGAAGG-3`; OLM1,
5`-AACTGACAGAGTGCCAGCTG-GCCAAGGACAGTGGAAAATC-3`; OL
M2,
5`-CAGCCACATCCTTCT-CTGATAACCAGCCTG-3`; OL
M3,
5`-TCGTGAGATACCTGA-AGAAGGCAGAACGCA-3`; OLC909-S,
5`-CTGTCAATGACTCCCATGCAGAAATA-3`; OLH910-Y,
5`-GTCAATGACTGCTATGCAGAAATAATC-3`; OLE912-A,
5`-ACTGCCATGCAGCAATAATCTCCCG3-`; OLH958-Y,
5`-GACTGTGTCATTCTATCTGTATATCAG-3`; OLC966-S,
5`-CACTGCTCCGTCTGGAGATGGCG-3`; OLC1036-S,
5`-GTACCATGTCCTCTAGTGACAAAATC-3`; and OLA1051-T,
5`-GGCCTGCAAGGGGCACTGTTGACCC-3`. The mutated bases are underlined. The
junction sites for the deletion mutants are indicated by hyphens. For
construction of pVL-F-DRADA140, two polymerase chain reaction (PCR)
primers were used: FLAGUP,
5`-ATAAGAATGCGGCCGCTAAACATGGCTGACTACAAGGACGACGATGACAAGATGAATCCGCGGCAGGGG-3`,
and FLAGDW, 5`-ATCATACGAGGTCACTGTCAGATCTGC-3`. Restriction sites
(underlined) for NotI for the sense primer (FLAGUP) and XbaI for the antisense primer (FLAGDW) were included in the 5`
end.
Deletion and Site-directed Mutagenesis
A
recombinant construct that coded for a full-length DRADA (pVL-DRADA140)
was used as a starting plasmid(21) . The total size of the
plasmid is about 14 kb with 4.5 kb of cDNA containing the entire
translated region of DRADA as an insert in pVL1393 (Pharmingen, San
Diego, CA). For preparation of C1, 2, 3, and 4, the wild-type
plasmid was subjected to deletion mutagenesis around the putative
C-terminal deaminase catalytic domain (amino acids 835-1221) using ExoIII exonuclease (33) after cleavage at the StuI site located near the C-terminal end. The linearized DNA
was incubated with ExoIII nuclease (New England Biolabs Inc.)
for various times in order to generate nested deletions. The reaction
products were then treated with mung bean nuclease (Boehringer
Mannheim) to trim the ends of the ExoIII-treated plasmid DNAs.
Inserts with various sizes of deletions were released by restriction
digestion at the SstI site located between two DRBMs. The
digestion products were fractionated on a 1.5% agarose gel, and the
purified fragments were subcloned back into the SstI and StuI sites of the wild-type plasmid. The resultant C-terminal
deletion clones were sequenced through the junction sites, using the
373A DNA sequencing system (Applied Biosystems, Foster City, CA). The
DNA sequences obtained were analyzed by the BESTFIT program of the
University of Wisconsin Genetics Computer Group (GCG) sequence analysis
software (version 7.0)(34) . Only in-frame deletions were
selected for further experiments.
,
and 10 clones each of the resultant transformants were screened for the
absence of the StuI site. The candidate mutant clones were
sequenced using Sequenase version 2.0 (U. S. Biochemical Corp.) to
verify the mutation site. All point mutation constructs were designated
as C909-S, H910-Y, E912-A, H958-Y, C966-S, C1036-S, and A1051-T in
accordance with the names of the mutagenic oligonucleotide.
M1,
M2,
and
M3, were achieved as described above using the same kit and
selection oligo. The mutagenic oligos were designed in such a way that
each has 15-20 bases of homology to both flanking sequences of
the deletion. The precise deletions were confirmed by sequencing.
Double DRBM deletion mutants
M1M3 and
M2M3 were reconstructed
from single deletion mutants using a unique restriction site AflII located near the NH
-terminal and SstI located between DRBM2 and DRBM3. The AflII-SstI fragments of
M1 and
M2 were
subcloned into
M3 at AflII and SstI sites,
respectively, resulting in double DRBM deletions of
M1M3 and
M2M3.
-terminal of the full-length DRADA
wild-type protein was constructed as follows: the entire coding region
of DRADA was amplified using a plasmid pVL-DRADA140 (21) and a
set of PCR primers FLAGUP and FLAGDW. This PCR procedure created a
methionine residue followed by the polypeptide sequence,
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG epitope-tag), at the 5` of the
NH
-terminal of the authentic DRADA140 protein. In addition,
it also removed the GC-rich 5`-untranslated region and long
3`-untranslated region, and created a new Kozak (36) sequence
that is strongly preferred by baculovirus for protein translation
initiation (37) at the new NH
-terminal region. The
PCR products were digested with NotI and XbaI, and
the 3.9 kb NotI/XbaI DNA fragment was gel purified,
and then ligated into NotI and XbaI sites of pVL1392
vector (InVitrogen, San Diego, CA). DRBM deletions with FLAG epitope,
pVL-F-
M1, pVL-F-
M2, and pVL-F-
M3 plasmids, were prepared
by excising the AflII-XbaI fragments (3.7 kb)
containing deletions from the original mutant constructs and ligating
them into the AflII and XbaI sites of pVL-F-DRADA140.
A point mutation with FLAG epitope, pVL-F-E912-A, was created in the
same way as other point mutations except that pVL-F-DRADA140 was used
as a starting plasmid. The DRADA proteins containing the FLAG
epitope-tag prepared from these constructs were purified by a
monoclonal antibody M2 affinity column chromatography (Kodak).
Cell Culture
Spodoptera frugiperdera (Sf9) cells were obtained from The Wistar Institute Expression
Core Facility. The insect medium TMN-FH with 10% fetal bovine serum was
purchased from Pharmingen, and supplemented with 0.1% Pluronic-F68
(Life Technologies, Inc.), and 1% antibiotic-antimycotic (Life
Technologies, Inc.).
Recombinant Baculovirus
To obtain recombinant
baculovirus expressing human DRADA proteins, 2 10
Sf9 cells were co-transfected with a mixture of 3 µg of
recombinant plasmid and 2.5 µg of BaculoGold linearized baculovirus
DNA following the manufacturer's instructions (Pharmingen). The
transfected cells were incubated at 27 °C for 5 days. Primary
recombinant viruses collected as supernatant of the culture were then
reamplified by infecting 1
10
Sf9 cells with 500
µl of primary recombinant virus stocks. Amplified viruses were
collected after 3 days of incubation at 27 °C.
Virus Infection and Cell Extract
Preparation
Approximately 1 10
Sf9 cells
were infected with 500 µl of amplified recombinant virus, incubated
at 27 °C for 48 h. The infected cells were collected and pelleted
by centrifugation. The infected cells were washed once with 5 ml of
ice-cold phosphate-buffered saline without CaCl
or
MgCl
and 1 ml of TEN (0.15 M NaCl, 40 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol (DTT), and 1
proteinase inhibitor mix), then resuspended in 150 µl of 0.25 M Tris-Cl, pH 7.8, containing 1 mM DTT and 1
proteinase inhibitor mix. Following sonication, the cell debris was
pelleted at 4 °C by centrifugation. The supernatant was mixed with
an equal volume of buffer containing 0.1 M Tris-Cl, pH 7.5,
0.2 M NaCl, 10 mM EDTA, 40% glycerol, 2 mM DTT, and 2
proteinase inhibitor mix. The proteinase
inhibitor mix (500
) was made with phenylmethylsulfonyl fluoride
(0.2 M), pepstatin (1.5 mM), leupeptin (4.2
mM), and aprotinin (0.3 mM). For each construct,
three to six independent infections and cell extract preparations were
carried out in order to obtain a reliable quantitative estimate for the
mutated DRADA enzyme activity.
In Vivo Labeling of Recombinant DRADA Protein
The
production of recombinant DRADA protein was assessed by
[S]methionine labeling of infected
cells(37) . Sf9 cells (2
10
) were infected
with 500 µl of each amplified recombinant virus stock at
multiplicity of 10-20 for 46 h. The infected cells were collected
and resuspended in 1 ml of methionine-free Grace's medium (Life
Technologies, Inc.) with 10% heat-inactivated fetal bovine serum.
Following incubation at 27 °C for 1 h, the medium was replaced with
1 ml of fresh methionine-free medium containing 40 µCi of
Tran
S-label (ICN Biomedicals, Costa Mesa, CA) and
incubated for another 1 h. The cells were washed with 1 ml of
phosphate-buffered saline three times. The labeled protein was analyzed
by SDS-polyacrylamide gel electrophoresis (PAGE) (8%) and fluorography.
Purification of Recombinant DRADA Proteins
The
recombinant DRADA proteins, F-DRADA140, F-DRADAM1, F-DRADA
M2,
F-DRADA
M3, and F-DRADAE912-A containing a FLAG epitope-tag were
purified on an anti-FLAG M2 immunoaffinity gel column. Approximately 20
mg of crude extract protein made from Sf9 cells infected with various
recombinant baculoviruses were applied to 1 ml of anti-FLAG M2 affinity
gel (Eastman) equilibrated with buffer A (0.05 M Tris, pH 7.0,
0.05% Nonidet P-40, 20% (v/v) glycerol, and 1 mM of
-mercaptoethonol plus 0.15 M NaCl). The flow-through
fraction was applied two more times to the same column to increase the
binding efficiency of the recombinant protein. The column was washed
with 30 ml of buffer A containing 0.15 M NaCl, then with 30 ml
of buffer A containing 0.75 M NaCl and once again with 30 ml
of buffer A containing 0.15 M NaCl. The recombinant DRADA
protein was extracted with 6 ml of buffer A containing 75 µg/ml of
FLAG peptide. The eluted proteins were pooled and concentrated by
Centricon 30 (Amicon, Beverly, MA) and stored at -70 °C.
Protein Assay
Protein concentration was determined
using the Bio-Rad protein assay reagent or the integrated separation
systems protein-gold assay system with bovine serum albumin as a
standard. The purified proteins were analyzed by one-dimensional
SDS-PAGE (4% acrylamide stacking gel, 8% acrylamide resolving
gel)(38) . Protein bands were visualized by silver
staining(39) .
DRADA Assay
The DRADA enzymatic activity was
assayed in vitro(4, 40) . Unless specified
otherwise, the reaction was carried out in 100 µl, which contained
10 fmol of [-
P]ATP-labeled c-myc dsRNA, 0.05 mM Tris (pH 7.0), 0.2 M NaCl, 5
mM EDTA, 1 mM DTT, 20% glycerol, and various amounts
of cell extract or purified proteins. After incubation at 37 °C,
the reaction products were deproteinized and then precipitated with
ethanol as described previously. The DRADA reaction products were
digested with nuclease P
into 5`-mononucleotides and
analyzed by one-dimensional TLC. The solvent system used was 0.1 M sodium phosphate, pH 6.8, ammonium sulfate, 1-propanol, 100:60:2
(v/w/v), as described previously(41) . The radioactivity of the
adenosine and inosine spots on TLC plates was quantified by a
PhosphorImaging system (Molecular Dynamics, Sunnyvale, CA).
RNA-binding Assay
A filter-binding assay (42) was carried out by using recombinant DRADA proteins
containing the FLAG epitope-tag at their NH-terminal and
[
-
P]ATP-labeled c-myc dsRNA (575
bp), or c-myc antisense single-stranded RNA (ssRNA) (605
nucleotides)(4) . Each reaction mixture, kept on ice, contained
10 ng of DRADA and varying quantities of dsRNA or ssRNA (0.05-5
nM) in 100 µl of the DRADA assay buffer containing 0.05 M Tris (pH 7.0), 0.1 M NaCl, 5 mM EDTA, 1
mM DTT, 20% glycerol, 5 µg/ml E. coli tRNA, and 5
µg/ml bovine serum albumin. The reactions were incubated for 10 min
at 37 °C and immediately filtered through a nitrocellulose membrane
(25 mm and 0.45 µm; Millipore, Bedford, MA) on a manifold
filtration apparatus (Schleicher & Schuell) at a slow flow rate.
The membranes were subsequently washed five times with 0.5 ml each of
the ice-cold DRADA assay buffer, dried, and subjected to liquid
scintillation counting.
Identification of Four Critical Residues Likely to Be
Involved in Zinc Chelation and Proton Transfer of DRADA
We
aligned the human DRADA sequence with other known deaminases and with a
recently identified nematode gene, T20H4.4(43) , that has a
high degree of homology to DRADA and is postulated to be a prototype of
the non-vertebrate DRADA gene(21) . We found that several short
stretches of contiguous amino acid residues, including two tripeptide
sequences HAE and PCG located in the C-terminal region, are highly
conserved (Fig. 1). We believe these conserved amino acid
residues are directly involved in the catalytic mechanism of
DRADA(21) . Among these residues, of special interest are the
amino acids histidine and cysteine, which can participate in
coordination of a zinc atom and formation of a catalytic center, as was
predicted by examination of the three-dimensional structure of
adenosine deaminase (44) and cytidine deaminase(45) .
We first prepared four serial deletion mutations in the C-terminal
region as recombinant baculovirus DRADA C1,
C2,
C3, and
C4 (Fig. 1). The mutated DRADA proteins were expressed as
recombinant baculovirus in insect cells (Fig. 2A), and
the A to I conversion activity of altered enzymes were tested in
vitro using crude extract proteins made from Sf9 cells infected
with four different DRADA mutant constructs (Fig. 2B).
Although host Sf9 cells also contain a trace amount of the DRADA
activity (0.2% A to I conversion in one experiment), its very low level
did not interfere with our quantitation of the enzymatic activity for
each mutant protein. We found that all of the C-terminal deletion
mutants lost enzyme activity (Fig. 2B), probably due to
a drastic change in the folding of the enzyme structure. Therefore, for
mutagenesis of this putative catalytic domain, site-directed point
mutations at some of the conserved amino acid residues were introduced (Fig. 1). Based on alignment between human DRADA and
T20H4.4(43) , all histidines (His
and
His
) and cysteines (Cys
, Cys
,
and Cys
) found in the regions conserved between these
two genes were mutated to conservative replacements, tyrosine and
serine, respectively. Among these residues, His
and
Cys
are within the two tripeptide sequences highly
conserved among different cytidine and deoxycytidylate
deaminases(30, 31, 32, 46) . These
residues are known to be involved in zinc-chelation at least in the
case of cytidine deaminase(45) , whereas Cys
,
His
, and Cys
were targeted as the
potential third and fourth zinc-coordinating amino acid residues.
M1,
M2,
M3,
M1M3, and
M2M3) and the deaminase catalytic domain
(
C1-
C4) deletion mutants. The numbers below each line
indicate the amino acid residues present in the deletion mutant
proteins. In the lower panel, the residues within the deaminase
catalytic domain changed by site-directed mutagenesis are indicated by arrows below the mutated wild-type residues. The sequence of
15 short stretches of contiguous amino acid residues, found to be
conserved between the human DRADA and a nematode gene T20H4.4 and
proposed to form the deaminase catalytic domain (amino acids 889-1160),
is shown. The two tripeptide sequences, HAE and PCG, which are highly
conserved among deaminases and predicted to participate in the
formation of the catalytic center, are highlighted. A summary
of the DRADA enzymatic activity (A to I conversion) of each mutant
observed is indicated at right or bottom. The DRADA
activity is coded in the range +++ to +, where
+++ is wild-type activity and + indicates a low but
significantly higher than background level of
activity.
S]methionine, and proteins
were fractionated by SDS-8% polyacrylamide gel electrophoresis. In
addition to the expected size band, the
C1 construct produced
discrete lower molecular weight protein bands which may be the products
of proteolysis of the full-length mutant protein. B, DRADA
activity for the C-terminal deletion mutants was determined by a base
modification assay. The 100-µl reaction contained 50 µg of
extract proteins and 10 fmol of
[
-
P]ATP-labeled c-myc dsRNA in the
standard assay buffer (see ``Experimental Procedures'').
Following incubation for 1 h at 37 °C, the reaction product was
deproteinized and digested with P
nuclease. The digests
were analyzed by one-dimensional TLC. The relative positions for
inosine 5`-monophosphate (pI) and adenosine 5`-monophosphate (pA) are indicated. Inosines converted from adenosines were
estimated by quantitating the ratio of pI and pA spots on TLC plates
with the PhosphorImaging system (Molecular
Dynamics).
We
found that substitutions at His and Cys
abolished the enzyme activity completely, whereas DRADA mutants
with alterations at Cys
and His
still
exhibited some enzyme activity (32 and 7% of the wild-type,
respectively), suggesting the critical importance of His
and Cys
in zinc coordination (Fig. 3).
Substitutions of Cys
also abolished the enzyme activity,
and, therefore, Cys
residue may act as the third protein
ligand of the zinc, together with His
and
Cys
. Cys
and His
may also
play important roles for the DRADA catalytic mechanism, but probably
not as zinc-chelating ligands.
Glu, which is expected
from the three-dimensional structure of E. coli cytidine
deaminase (45) to subserve the critical proton transfer
function of the enzyme, was changed to alanine. The DRADA mutant E912-A
with a Glu
Ala substitution exhibited no enzyme
activity (Fig. 3). Mutant A1051-T retained activity almost equal
to that of wild-type (Fig. 3).
The First and Third DRBM Repeats Are Critical for DRADA
Catalytic Activity
The three repeats of DRBM present in the
central region of DRADA are predicted to serve as a substrate binding
domain for this enzyme(21) , which has a strict specificity for
dsRNAs(1, 2, 5) . We confirmed this region as
the dsRNA binding domain by expressing this region containing only the
three DRBMs as a glutathione S-transferase fusion protein in E. coli and testing it by the filter binding assay. The
glutathione S-transferase-3DRBM truncated DRADA protein
exhibited a strong affinity for dsRNA (data not shown). In order to
examine the roles played by these motifs in the DRADA enzyme activity,
we then generated five deletion mutants in which each of the three
DRBMs, or a combination of two DRBMs was deleted from the intact
wild-type DRADA ( Fig. 1and Fig. 4A). We assayed
these DRBM mutants for their enzyme activity, and found that two
deletion mutants M1M3 and
M2M3, which have only a single
DRBM, lost activity completely (Fig. 4B), suggesting
that the DRADA enzymatic activity requires more than one DRBM. In
contrast to the results obtained with single DRBM mutants, unexpected
results were obtained with the DRBM mutants in which only one of the
three DRBMs had been eliminated. While the mutants
M1 and
M3
exhibited, if any, very little activity, the mutant
M2 retained
enzymatic activity comparable to that of wild type (Fig. 4B). These results suggest not only that DRADA
requires at least two DRBMs, but also that the first and third repeats
may provide indispensable functions for its enzymatic action. This
result, indicating the nonequivalent nature of the three DRBMs, is
surprising in light of the highly conserved amino acid sequences of the
three DRBMs(21) , and also the notion that the DRBMs found in
other dsRNA-binding proteins may be equivalent to each
other(28, 29) .
The Expression Levels of Mutant Proteins
In
vivo labeling and analysis of the recombinant DRADA proteins by
SDS-PAGE confirmed that, in general, the mutant proteins and the
wild-type protein were present at similar levels in the infected Sf9
cells (Figs. 2A, 3A, and 4A). All deletion
mutants showed a decreased size as expected. For certain mutants,
especially C909-S, the decreased protein level could be a factor in the
reduced activity of crude extracts of Sf9 cells (32% of wild-type
level). However, the complete loss of the enzymatic activity observed
with H910-Y, E912-A, C966-S, and some deletion mutants cannot be due to
a reduced protein level. These mutants did not show any A to I
conversion activity even after increase of the extract proteins or the
incubation time for the enzyme assay (data not shown). In addition, Sf9
cells were infected three to six times separately with individual
recombinant viruses at various multiplicity. The base modification
assays were carried out with cell extracts made from these separate
infections. We found that the relative DRADA activities among wild-type
and mutants, M2, C909-S, H958-Y, and A1051-T, were very similar
(within ± 20%) among different extracts (data not shown). The
complete loss or dramatic decrease of the DRADA activities for H910-Y,
E912-A, C966-S,
M1,
M3,
M1M3,
M2M3, and all of
C-terminal region deletion mutants was also confirmed by separate
infection experiments. Thus, the total loss of the enzyme activity
observed with certain mutants cannot be attributed to variation of
virus infection efficiency nor a decrease in the levels of their
protein expression in the Sf9 host cells.
Binding of DRBM Deletion Mutants to dsRNA
Because
our results with DRBM mutants raised the possibility that each of the
three DRBMs may have significantly different functions in the DRADA
reaction, we next examined binding of these DRBM deletion mutants to
dsRNA using purified recombinant DRADA proteins, a synthetic c-myc dsRNA (575 bp), and a nitrocellulose filter binding assay
traditionally used for quantitation of interactions of nucleic acid
binding proteins with K stronger than 1
nM(42) . This assay was used previously to determine
the substrate dissociation constant of wild-type DRADA purified from
bovine liver, which turned out to have a much higher affinity for
substrate than other deaminases (40) .
-terminal
end were prepared from original constructs DRADA-140 (WT),
M1,
M2, and
M3 as well as the catalytic domain mutant
E912-A. The recombinant FLAG-DRADA fusion proteins expressed in
infected Sf9 cells were purified using the FLAG epitope by anti-FLAG M2
antibody affinity column chromatography (Fig. 5B). The
binding of all DRADA mutants maintained specific binding to dsRNA, and
did not bind to ssRNA (Fig. 5A). The affinity constant
was obtained by Scatchard-type analysis (Fig. 5A). The
reverse Scatchard plots for wild-type and all mutant DRADA proteins
tested were almost superimposable and, therefore, plots for only
wild-type DRADA,
M1, and
M2 are shown in Fig. 5. The K
, derived from the intercept on the
abscissa of the Scatchard plot, was identical (0.11 nM) for
wild-type and all four of the mutant DRADA proteins. It is of special
interest that DRBM deletion mutants F-
M1, F-
M2, and F-
M3
all have K
values identical to that of
the wild-type protein. This shows that the dramatically decreased DRADA
enzymatic activity observed with
M1 and
M3 is not due to
their altered dsRNA binding capacity.
-
P]ATP labeled c-myc dsRNA (575
bp) or antisense ssRNA (nt 605) was analyzed by a nitrocellulose
binding assay. Ten ng of purified FLAG-DRADA fusion protein for each
mutant were incubated in duplicate with various concentrations of dsRNA
or ssRNA, as described under ``Experimental Procedures.'' The
reverse Scatchard plots of c-myc dsRNA binding to wild-type
and various DRADA mutants were obtained. Since the plots for all
mutants analyzed were almost identical to that of wild-type DRADA,
plots for only wild type,
M1, and
M2 are shown. The intercept
at the abscissa defines the K, which was 0.11 for
wild type and all four mutants (
M1,
M2,
M3, and E912-A). B, the purified recombinant DRADA proteins used in this study
were examined by SDS-PAGE and silver
staining.
The Catalytic Mechanism of the DRADA
Reaction
Site-directed mutagenesis of His and
Cys
completely abolished the enzyme activity of the
mutated DRADA proteins, despite the fact that both amino residues were
substituted by conservative replacements. This result reinforced our
hypothesis that these two amino acid residues, both located within the
tripeptide sequence HAE and PCG conserved among different cytidine and
deoxycytidylate deaminase family
members(31, 32, 45, 46) , play a
critical role for the DRADA catalytic mechanism. Although the presence
of a zinc atom within DRADA has not yet been demonstrated directly, the
presence of short stretches of ``deaminase'' consensus
tripeptide sequences strongly suggests that these two residues
participate in zinc chelation. The recent x-ray crystallography of E. coli cytidine deaminase confirmed the postulated
zinc-chelation site for that enzyme(45) .
also resulted in the loss of enzyme activity. Since the
coordination of the zinc atom requires at least three protein ligands
for both adenosine deaminase (44) and cytidine
deaminase(45) , it is reasonable to postulate that this
Cys
residue is also involved in zinc chelation. Our
site-directed mutagenesis of Glu
confirmed the importance
of this residue for the enzymatic action. It most likely plays a
critical role in proton transfer functions required for the deamination
reaction, predicted once again by analogy from the three-dimensional
structure of E. coli cytidine deaminase(45) .
Interestingly, a similar mutation for the cytidine deaminase subunit
(REPR) of apolipoprotein B100 RNA editing enzyme has also been reported
to abolish the enzymatic activity (47) . A1051-T represents an
example of a residue which can tolerate a conservative substitution and
maintain almost full activity of the DRADA enzyme (Fig. 3), even
though Ala
is one of the amino acid residues conserved
between the catalytic domain of DRADA and T20H4.4(21) . The
result suggests that some of the conserved residues such as His
and Glu
are absolutely necessary for the enzyme
function, while others contribute only to the maintenance of the
overall protein folding and structure, and accommodate conservative
sequence alterations. Obviously, the final functional assignment of
amino acid residues shown as indispensable in this study must wait the
structural studies of DRADA complexed with dsRNA.
Distinctive Functions of the Three DRBMs in the DRADA
Enzyme Action
The DRBM is approximately 70 amino acid residues.
It may be divided into a less conserved NH-terminal
two-thirds and a more conserved C-terminal region. The C-terminal
region enriched for positively charged amino acids, especially
arginine, and predicted to form an
-helix, may interact directly
with the dsRNA helix structure(26, 28) . This new type
of RNA binding motif is found in a growing family of
proteins(24, 25, 26, 27, 28, 29, 47) .
The DRBM elements of these proteins have already been tested for their
dsRNA binding capacity with recombinantly expressed
DRBMs(23, 24, 26, 27, 28, 29) .
It appears that each DRBM is usually capable of binding to the dsRNA
independently(28, 29) , except in certain cases where
at least two repeats were needed for efficient
binding(25, 26) . There are two different types of
DRBM, a full-length long repeat and a short repeat(29) . Some
previously sequenced DRBMs have only the C-terminal
-helix region,
which led to the hypothesis that the N-terminal two-thirds of the motif
are less important for dsRNA binding(29) . DRADA contains three
full-length DRBM repeats(21) .
-helix forming region of the two DRBMs
on the same side of the RNA duplex. Such flexible interaction and
orientation of the two DRBMs may be necessary for strong binding
affinity of DRADA to dsRNA(5, 40) . Because of the way
the deletion was made, the DRBM deletion mutant
M2 contains a
longer spacer region (71 amino acids) between the remaining DRBM1 and
DRBM3, which may give a similar spacer effect of the DRBM2 played in
the wild-type enzyme. However, if this is the case, one may expect
significant differences in the dsRNA binding affinity between wild-type
or
M2 and
M1 or
M3. In contrast, K
values for all three DRBM deletion mutants were nearly the
same as that of wild-type DRADA protein, ruling out a simple spacer
effect.
-helix(26, 28) , is
indicated. Certain amino acid residues, part of the consensus sequence,
and present only in DRBM1 and DRBM3 but missing from DRBM2, are
indicated by arrowheads.
By comparing the amino acid sequences for the three DRBM, we
noticed that there are several amino acid residues of the DRBM
consensus sequence present in DRBM1 and DRBM3 but missing in DRBM2 (Fig. 6). In addition, we recently compared the amino acid
sequence of human and mouse DRADA deduced from cDNA
sequences.(
)Although all three DRBM sequences
are highly conserved between human and mouse DRADA, DRBM1 and DRBM3
contain 10 and 9 amino acid residue extensions of evolutionarily
conserved sequence upstream of the N-terminal boundary of the 72-amino
acid motif, whereas DRBM2 does not contain this 5` extension.
The evolutionarily conserved sequence present upstream of DRBM1
and DRBM3 may add to these two repeats additional functions other than
dsRNA binding, which distinguishes them from DRBM2.
Separate Functional Domains of DRADA
As we
predicted previously(21) , results from the present studies
suggest that DRADA indeed contains at least two separate functional
domains: a substrate binding domain consisting of three repeats of DRBM
and the C-terminal region consisting of the catalytic or deaminase
domain. These two separate functional domains likely interact with each
other, especially if adenosine residues to be deaminated are located
within the dsRNA region to which the binding domain binds. As already
mentioned in the previous section, some DRBM repeats may play active
roles in the deamination process. DRADA seems to be self-contained,
with all of the necessary functional domains within it, and
recombinantly expressed and purified enzyme is indeed capable of
carrying out deamination of multiple adenosines to inosines in
vitro by itself. This structural feature of DRADA is in contrast
to that of the recently cloned REPR, the cytidine deaminase component
of the multisubunit complex (editosome) for editing of apoliprotein
B100 RNA(46) . In this editing system, the substrate binding
subunit, which recognizes a specific cis-RNA element termed
the ``mooring sequence'' essential for precise editing, seems
to be required in addition to REPR (52) . Moreover, an
additional auxiliary factor may also be required for REPR to carry out
precise editing of apoliprotein B100 RNAs(46, 52) . In
view of the high degree of precision necessary for selecting specific
adenosine residues at the GluR RNA editing
sites(10, 11, 12, 18) , the lack of
sequence specificity of DRADA has been a
puzzle(7, 8, 18) . It remains possible that
the apparently self-contained enzyme, DRADA, may yet require additional
factor(s) for site-selective editing of natural substrate RNAs such as
GluR transcripts in vivo. DRADA mutants generated in this
study are likely to be useful for our future studies of RNA editing by
DRADA.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.