From the Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and § Boston University, Biomolecular Engineering Research Center, Boston, Massachusetts 02111
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ABSTRACT |
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The subunit of the heterotrimeric
G proteins that transduce signals across the plasma membrane is made up
of an amino-terminal
-helical segment followed by seven repeating
units called WD (Trp-Asp) repeats that occur in about 140 different
proteins. The seven WD repeats in G
, the only WD repeat protein
whose crystal structure is known, form seven antiparallel
sheets
making up the blades of a toroidal propeller structure (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner,
B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell
83, 1047-1058; Sondek, J., Bohm, A., Lambright, D. G., Hamm,
H. E., and Sigler, P. B. (1996) Nature 379, 369-374). It is likely that all proteins with WD repeats form a
propeller structure. Alignment of the sequence of 918 unique WD repeats
reveals that 85% of the repeats have an aspartic acid (D) residue (not
the D of WD) in the turn connecting
strands b and c of each
putative propeller blade. We mutated each of these conserved Asp
residues to Gly individually and in pairs in G
and in Sec13, a yeast
WD repeat protein involved in vesicular traffic, and then analyzed the
ability of the mutant proteins to fold in vitro and in
COS-7 cells. In vitro, most single mutant G
subunits
fold into G
dimers more slowly than wild type to a degree that
varies with the blade. In contrast, all single mutants form normal
amounts of G
in COS-7 cells, although some dimers show subtle
local distortions of structure. Most double mutants assemble poorly in
both systems. We conclude that the conserved Asp residues are not
equivalent and not all are essential for the folding of the propeller
structure. Some may affect the folding pathway or the affinity for
chaperonins. Mutations of the conserved Asp in Sec13 affect folding
equally in vitro and in COS-7 cells. The repeats that most
affected folding were not at the same position in Sec13 and G
. Our
finding, both in G
and in Sec13, that no mutation of the conserved
Asp entirely prevents folding suggests that there is no obligatory
folding order for each repeat and that the folding order is probably
not the same for different WD repeat proteins, or even necessarily
constant for the same protein.
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INTRODUCTION |
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The subunit of the heterotrimeric G proteins that transduce
signals across the plasma membrane is made up of two distinct regions
as follows: an amino-terminal
-helical segment, followed by 7 repeating units called WD repeats that occur in about 140 different
proteins (reviewed in Refs. 1 and 2). Members of the family of WD
repeat proteins do not have an immediately obvious common function but
are involved in diverse cellular pathways such as signal transduction,
pre-mRNA splicing, transcriptional regulation, cytoskeletal
assembly, and vesicular traffic (2).
Each WD repeat consists of a conserved core of approximately 40 amino
acids (typically bracketed by the dipeptides GH (glycine-histidine) and
WD (tryptophan-aspartic acid)) and a variable region of 7-11 amino
acids (2). G is the only WD repeat protein whose crystal structure
is known (3-5). The seven WD repeats in G
are arranged in a ring to
form a propeller structure with seven blades. Each blade of the
propeller consists of a four-stranded antiparallel
sheet oriented
so that the outer surfaces of the torus are composed of the sheet
edges, whereas the turns protrude from the two flat surfaces (see Fig.
1). It is likely that all proteins with WD repeats form a propeller
structure, although with varying numbers of blades corresponding to
varying numbers of repeating units. WD repeats are not essential to
form a propeller. Other families of proteins with no sequence
similarity to WD repeat proteins form propellers whose blades are
virtually identical to those in G
(reviewed in Ref. 6).
Nevertheless, within the subset of propellers formed of WD repeats, it
is reasonable to suppose that the most highly conserved residues play
an important role either in the function or the structure.
The WD repeats are not characterized by a rigidly conserved sequence
but rather by their fit to a regular expression that allows limited
variation at each position (2). However, alignment of the sequences of
918 unique WD repeats in our data set reveals that one residue is the
most conserved; an aspartic acid residue (D, not the D in WD) located
in the loop connecting strands b and c of each propeller blade in
G
(and presumably in all other WD repeat proteins) occurs in 85% of
the repeats. In another 9%, the residue is Glu or Asn. This
extraordinary conservation suggests that the Asp residue performs an
important function that is shared by all WD repeats. Since the WD
repeat proteins do not appear to bind to any common molecule, we tested
the hypothesis that the conserved Asp plays a role in the folding of
the propeller.
The occurrence of a conserved residue at an equivalent position in each repeat allowed us to ask a number of questions. Are all the Asp residues equivalent within a protein? Are the consequences of mutating Asp to Gly the same in different proteins? It is not known whether the WD repeat or other propeller proteins fold by a single or multiple pathways. If there is a single pathway, we would expect that mutation of a critical Asp would have a large effect on folding kinetics, whereas if multiple pathways to the final structure exist, a single mutation might have little effect since it would be kinetically less important if an alternative pathway could be followed (7).
To analyze such questions, we mutated the conserved Asp to Gly in two
WD repeat proteins, G and Sec13, a yeast protein involved in
vesicular traffic (8). Mutations were inserted one at a time or two at
a time. We mutated Asp to Gly because a Gly residue makes the
polypeptide chain flexible and is compatible with formation of a turn.
Futhermore, the side chain of Asp points into the structure of
and,
in some cases, makes contact with other residues within the propeller
blade (see "Discussion"). Therefore, we wanted an amino acid that
had a small side chain not to confound interpretation by effects
produced by the side chain of the amino acid substituted for the
aspartic acid residue. G
was chosen because its crystal structure is
known. Sec13 has 6 repeats and no amino- or carboxyl-terminal extension. We have made and tested a model of Sec13 based on the structure of G
(9). The model predicts that the conserved Asp are in
equivalent positions to G
. The G
and Sec13 differ in their
requirements for folding. G
cannot fold completely without G
(10)
to which it is very tightly bound in the native structure. Furthermore,
folding and/or assembly probably requires as yet undefined chaperones
(11). In contrast, Sec13 can fold into a globular, trypsin-resistant
structure when synthesized in Escherichia coli, wheat germ,
rabbit reticulocyte lysate in in vitro translation systems,
or in mammalian cells (9, 12). If it requires chaperones at all, it can
productively interact with several different ones.
We have analyzed the ability of G to fold and assemble with G
and
of Sec13 to form a compact structure after synthesis in vitro and in COS-7 cells. This comparison allows us to
discriminate between mutations that affect the end state and those that
affect the rate of folding.
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EXPERIMENTAL PROCEDURES |
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Cell Culture, Transfection, and Biosynthetic Labeling-- COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS),1 2 mM glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin. Transfections were done with LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. Typically, cells on 6-well dishes were transfected with 2 µg of total DNA and 15 µg of LipofectAMINE in 1 ml of Opti-MEM (Life Technologies, Inc.) for 5-6 h, after which 1 volume of Opti-MEM supplemented with 8% FBS was added to each well. 18-24 h after the start of transfection, this medium was replaced with complete culture medium (Dulbecco's modified Eagle's medium + 10% FBS), and cells were incubated at 37 °C overnight and then biosynthetically labeled. For labeling, cells were first starved in a methionine/cysteine-deficient RPMI medium containing 5% dialyzed fetal bovine serum for 30-45 min and then labeled with 0.1 mCi of Express Protein Labeling Mix (NEN Life Science Products) per well (1 ml) in the presence of 10% dialyzed FBS. After 2.5-3 h at 37 °C, the medium was removed, and cells were washed twice with PBS and harvested by trypsinization.
Mutagenesis and Plasmid Construction--
Mutations in the 1
cDNA were generated using the Altered Sites in vitro
mutagenesis system (Promega). To construct a hexahistidine-tagged
1 (H
1) subunit, the initial methionine
was mutated to glutamine, and at the same time, a HindIII
and a PstI site were introduced. An annealed double-stranded
DNA encoding the first methionine and six histidines was synthesized
and ligated between the new HindIII site and the
EcoRI site from the pAlter vector. The amino acid sequence
of the amino-terminally tagged
1 is MSHHHHHHGSLLQ. In
addition, to facilitate the transfer of the mutants to other vectors, a
silent mutation corresponding to amino acids 144 and 145 was introduced
into
1 to create a unique KpnI site. This construct (H
1 in pAlter) was used as a template for
creating all mutants. The mutated residues were Asp-76 in repeat 1 (H
1[D1]), Asp-118 in repeat 2 [H
1[D2]), Asp-163 in repeat 3 (H
1[D3]), Asp-205 in repeat 4 (H
1[D4]), Asp-247 in repeat 5 (H
1[D5]), Asp-291 in repeat 6 (H
1[D6]), and Asp-333 in repeat 7 (H
1[D7]), and all were changed to glycine using the
codon that allowed a single base substitution (GGT or GGC). All
mutations were confirmed by double-stranded sequencing. For expression
in COS-7 cells, the wild-type (wt) H
1 cDNA or the
mutated forms were transferred to the pcDNA3 vector (Invitrogen).
The single mutants H
1[D2] and H
1[D3]
were obtained from the double mutant H
1[D2-3] by inserting a HindIII-KpnI fragment containing the
[D2] mutation or a KpnI-BamHI fragment with the
[D3] mutation into an H
1-pcDNA3 background.
Likewise, the double mutants H
1[D1-7] and
H
1[D2-7] were generated by inserting the
HindIII-KpnI fragment from either H
1[D1] or H
1[D2] into
H
1[D7] in pcDNA3. For H
1[D4-7],
H
1[D4] in pcDNA3 was cut with NdeI and
ligated into H
1[D7].
In Vitro Translation, Immunoprecipitation, and Trypsin
Digestion--
All proteins were transcribed and translated using the
TNT-coupled reticulocyte lysate system (Promega). Typically, 1 µg of plasmid DNA and 20 µCi of [35S]methionine or
[35S]cysteine were used in a 50-µl reaction. In all
cases, transcription was directed by the T7 promoter either from pAlter
or pcDNA3. To increase expression levels, all subunits were
subcloned into the PAGA-1 vector (provided by Dr. O. Reiner) that
contains a poly(A) sequence and the alfalfa mosaic virus leader
sequence, which has been previously shown to improve translation
efficiency (13). Synthesis of the desired product was routinely
verified by running 2-5 µl of the translation mixture in a small 11 or 13% polyacrylamide gel (14) followed by autoradiography with overnight exposure. Mixtures of independently translated
and
were then made, such that
was in excess and the subunits were incubated together at 37 °C for 90 min to dimerize. After
dimerization, the samples were either subjected to
immunoprecipitation or to trypsin digestion.
Immunoprecipitation from COS-7 Cells and ADP-Ribosylation-- Labeled cells were lysed in TNE buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA) plus 1% Triton X-100 and 0.25% deoxycholic acid supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, and 1 µg/ml each of soy and lima bean trypsin inhibitor). After lysis, all further steps were performed at 4 °C. The lysates (1 ml/well transfected cells) were first precleared with 50 µl of protein A-Sepharose slurry (50% v/v in PBS) for 45-60 min and then incubated for 2 h or overnight with 4 µl of 12CA5 monoclonal antibody (anti-HA epitope). At this point, each sample was usually split in 2 aliquots (500 µl each), and 40 µl of protein A-Sepharose slurry (1:1) was added to each one. After 45-60 min, samples were washed three or four times with the lysis buffer and once with 50 mM Tris-HCl, pH 8, 2 mM MgCl2, 1 mM EDTA (ADP-ribosylation buffer). One aliquot of each sample was then boiled in Laemmli sample buffer, and the other aliquot was subjected to ADP-ribosylation with Bordetella pertussis toxin. The reaction was carried out in a 30-µl volume containing 50 mM Tris-HCl, pH 8, 2 mM MgCl2, 1 mM EDTA, 10 mM dithiothreitol, 10 mM thymidine, 10 µM NAD, 1 mM NADP, 100 µM GTP, 1 mM ATP, 0.5 µCi of [32P]NAD, and 10 µg/ml activated pertussis toxin. After 30-60 min at 37 °C, the samples were washed with 1 ml of ice-cold ADP-ribosylation buffer, boiled in Laemmli sample buffer (14), and analyzed on 11% SDS-PAGE. For exposure of [32P] signal without contribution from [35S], a black film was placed between the gel and the film to be exposed.
Trypsin Digestion and Cross-linking of Samples from Transfected
Cells--
Lysis and immunoprecipitation were performed essentially as
described above, except that deoxycholic acid was not included in the
lysis buffer. Washes in the lysis buffer were followed by two
additional washes in 50 mM Tris-HCl, pH 7.5, and the final pellet of protein A-Sepharose beads was resuspended in 30 µl of this
same buffer. For trypsin digestion, 1 µl of 20 µM
L-1-tosylamido-2-phenylethylchloromethyl ketone-treated
trypsin (Cooper Biomed) was then added to one of the aliquots of each
sample (see above), and all samples were incubated at 30 °C for
10-15 min. The reaction was stopped with 2 µl of 100 mM
benzamidine. For cross-linking, 1 aliquot of each sample was treated
with 1.6 µl of freshly prepared 50 mM BMH
(1,6-bismaleimidohexane, Pierce) in Me2SO, and the other
aliquot received only Me2SO. After 20 min on ice, Laemmli
sample buffer containing 15% -mercaptoethanol was added, and the
samples were boiled for 5 min. The final products of both reactions
were resolved by SDS-PAGE on 11% polyacrylamide gels followed by
autoradiography.
Nickel Nitriloacetic Acid-Agarose Purification-- After labeling, transfected cells were lysed in buffer A (6 M guanidinium HCl, 0.1 M Na2HPO4/NaH2PO4, pH 8, 10 mM imidazole), and the lysate was then mixed with 50 µl of nickel nitriloacetic acid-agarose slurry (50% v/v in buffer A) (Qiagen) on a nutator for 3-5 h at room temperature. The beads were washed 5 times with buffer A and twice with 25 mM Tris-HCl, pH 6.8, 20 mM imidazole. Purified proteins were eluted by boiling the beads in Laemmli sample buffer supplemented with 200 mM imidazole and analyzed by SDS-PAGE followed by autoradiography.
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RESULTS |
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Mutation of 1--
Our goal was to determine if the
most conserved residue in the WD repeat family of proteins (the Asp in
the turn between
strands b and c of each blade structure, see Fig.
1) is essential for a WD repeat protein
to fold into a
-propeller. Mutations of aspartic acid to glycine
(Gly) were introduced in individual repeats of the
1
subunit, as well as in adjacent repeats (2, 3; 4, 5; 6, 7), or pairwise
in separate repeats (1, 7; 2, 7; 4, 7). All mutants were made in
1 tagged at the amino terminus with six histidine
residues (H
1), which was useful because it allowed us to
distinguish mutated from wild-type
in the transfection experiments
to be described below. There was no difference between H
1 and the wild-type
1 in any of the
assays used in this study (data not shown). The mutants are designated
by the number of the repeat in which the mutation is placed,
e.g. H
1 [D1], H
1 [D2],
etc.
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Analysis of 1 Mutants Synthesized in Vitro--
The
subunit does not fold into a compact structure without G
but,
instead, aggregates with itself and/or other proteins (10, 12).
dimers can be synthesized and assembled in vitro using
and
subunits synthesized in a rabbit reticulocyte lysate. Such
dimers are indistinguishable in their physical properties from
dimers purified from bovine brain (10). Indeed, we were able to
estimate the distance between the cysteine residues at the interface
between
and
by chemical cross-linking of in vitro
synthesized
to purified
. Our estimate was within 2 Å of
that subsequently found in the crystal structure (3, 5, 15).
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Analysis of the 1 Mutants Transiently Expressed in
COS-7 Cells--
The inefficiency of dimerization of G
and G
in
a rabbit reticulocyte lysate suggests that the missing or labile
components could be provided by a living cell. Therefore, we analyzed
the folding of mutant
1 subunits in COS-7 cells. The
function of transfected wild-type or mutated H
1 was
again assessed by immunoprecipitation of
by co-transfected HA-
2.
Because all
constructs were tagged with 6 histidines, we could
distinguish transfected
from endogenous
by the difference in
size (see Fig. 5). Therefore, we could always measure how much of the
subunit brought down by HA-
2 represented the transfected mutant protein. Fig. 5C and
Table I summarize the results of such experiments. Fig. 5 also shows the association of
with
that is discussed below. We
quantitated the intensity of the precipitated H
1 band
for each mutant and expressed it as a percentage of wild-type
H
1. In contrast to the results in the reticulocyte
lysate, there was no large difference in the amount of dimers
precipitated between wild type and any single mutant. Control
experiments verified that antibody was in excess in these experiments
(not shown).
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Interaction of the Mutant Dimers with the Subunit in
Transfected COS-7 Cells--
COS-7 cells were transfected with
wild-type or mutated H
1, HA-
2, and
i2. After labeling COS cells with
[35S]methionine, the H
1 mutants together
with any associated
subunit were immunoprecipitated through the HA
epitope on
2. In each experiment, we measured the amount
of
precipitated through the endogenous
that associated with
HA-
2 when no H
1 was transfected. Therefore, we could subtract the amount of
that could be accounted for by the small amount of endogenous
precipitated in each
experimental situation. This was always a very small correction (see
Fig. 5A). To verify that the strong 39-kDa band that
co-immunoprecipitated with
was indeed
, the immunoprecipitate
was treated with pertussis toxin and [32P]NAD (Fig.
5B). In every experiment, the intensity of the
35S-labeled
band was quantitated and related to the
amount of H
1 present in the immunoprecipitate (see Fig.
5D and Table I).
Trypsin Resistance of Mutant Dimers Transiently Expressed in
COS-7 Cells--
Immunoprecipitated
dimers derived from lysates
of [35S]methionine-labeled COS-7 cells were digested with
trypsin and analyzed on SDS-PAGE. The two bands corresponding to the
carboxyl- and amino-terminal fragments are indicated in Fig.
6A. Note that the amino-terminal fragment is bigger in mutant H
1 or
wild-type H
1 than in endogenous
because of the
hexahistidine tag. All the mutants, except H
1[D3] and
H
1[D7], gave an approximately normal amount of
fragments. The intensity of the carboxyl-terminal fragment band was
measured and normalized to the amount of H
1 found in the
undigested sample (see Fig. 6B and Table I). Consistent with the results from in vitro translated protein,
H
1[D3] formed dimers that were not resistant to
tryptic cleavage and were presumably not quite properly folded.
Nevertheless, H
1[D3] is able to bind
reasonably
well, confirming that the overall structure is close to native. Dimers
containing H
1[D7] were only partly resistant to
trypsin (45%), suggesting that the structure of the
-propeller must
be subtly different from that of the wild type. The only double mutants
that made a substantial amount of dimers included mutations in blades 3 and 7. Both produced little, if any, stable tryptic product.
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Cross-linking of Mutant Dimers Transiently Expressed in
COS-7 Cells--
The
subunit contains two structurally distinct
regions as follows: the seven WD repeats that give rise to the seven
bladed
-propeller, and an amino-terminal segment of approximately
20-30 amino acids that forms a coiled-coil with the amino terminus of the
subunit (3, 5). The trypsin digestion assay provides important
information about the integrity of the propeller itself. However, it
does not tell us if the amino-terminal
-helix, and hence the
coiled-coil, are properly formed. These aspects are better studied by
analyzing the ability of G
to be cross-linked to the
3 subunit using BMH. We have previously shown that this reagent specifically cross-links a cysteine residue in the
-helix of
1 (cysteine 25) to cysteine 30 in
3 (15). Because the
cross-linker has a very limited flexibility, this assay is very
sensitive to changes in the structure that might alter the distance
between these two residues. Wild-type and mutated H
1
subunits labeled with [35S]methionine were
co-immunoprecipitated through HA-tagged
3 (Fig. 7). BMH cross-linked wild-type and mutant
H
1 to
3 to give a major cross-linked
product of ~50 kDa corresponding to
H
1-HA
3 (3). The second, fainter band
observed right below corresponds to endogenous
subunits
cross-linked to HA-
3. This band becomes more evident
when H
1 is not included in the transfection (see 1st two lanes, labeled HA-
3). These
results indicate that the aspartic acid mutations do not induce changes
in the structure of the amino-terminal part of G
or in the relative
orientation of the G
subunit in that region. The fact that even the
mutants that showed an abnormal behavior on trypsin digestion (such as H
1[D3] or H
1[D7]) are cross-linked to
3 confirms that the
-propeller and the amino-terminal
extension are independent regions and that alterations in one do not
necessarily alter the conformation of the other.
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Effect of Aspartic Acid Mutations in Another WD Repeat Protein,
Sec13--
To determine if the results obtained with the G protein
1 subunit were generalizable to other members of the WD
repeat family, we undertook the same analysis on another WD repeat
protein, Sec13, a yeast protein involved in vesicular traffic. This
protein differs from G
in three important respects: it has six
repeats with no amino- or carboxyl-terminal extensions; it has no
-like partner protein; and it folds even in E. coli,
showing no requirement for mammalian over bacterial chaperonins (9). We
applied the same strategy as with
1, mutated the
conserved aspartic acid to glycine in each repeat and analyzed the
ability of the mutants to fold into a native structure. To facilitate
our subsequent analysis, mutations were introduced in a Sec13 construct
that had been previously tagged with an HA epitope at the amino
terminus (HA-Sec13). We know from our previous studies that Sec13
translated in vitro forms a globular, symmetric protein with
a Stokes radius of 26 Å. The compact structure is resistant to tryptic
cleavage, despite the presence of multiple (28) potential cleavage
sites throughout the sequence (12). We synthesized the mutant Sec13 proteins in a rabbit reticulocyte lysate and measured their Stokes radius and resistance to proteolysis by trypsin. All of the mutants were equally well translated, and all eluted from a calibrated AcA 34 column with a Stokes radius of 26-27 Å (data not shown, see Ref. 12
for an example of the elution pattern). The width of each peak at
half-height was the same for mutant and wild-type protein, indicating
that there was no detectable increased size heterogeneity in the
mutant. Therefore, none of the aspartic acid mutations prevented
folding in vitro. However, mutation of Asp in repeats 2, 3, 4, or 5 eliminated the resistance to tryptic cleavage (Fig.
8A). Most likely, these
mutants form a less rigid structure that allows some tryptic sites to
become exposed. Mutations in blades 1 and 6 were normal in both assays.
The double mutant, HA-Sec[D1-6], was also resistant to tryptic
cleavage (data not shown). The same results were obtained when the
HA-Sec13 mutants were transfected in COS-7 cells, immunoprecipitated
through the HA epitope, and digested with trypsin (see Fig.
8B and Table I). These experiments confirmed our previous
observation that not all of the conserved aspartic acids are
equivalent, and that each contributes to a different degree to the
stabilization of the
propeller. They further support the idea that
not all aspartic acids are essential for a WD protein to fold properly.
In addition, our results indicate that the effect of mutating one of
the conserved aspartic acids cannot be predicted based on the position
of the repeat in which that residue is found, because the location of the essential or non-essential aspartic acids is not conserved in
different WD repeat proteins.
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DISCUSSION |
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These studies show that no single mutation of a conserved aspartic
acid residue in G to a glycine prevents the formation of a compact
and substantially folded structure. Under optimal conditions
(e.g. in a mammalian cell) and given enough time, each of
the single mutant G
subunits can form a dimer with G
, bind G
,
allow the ADP-ribosylation of
by pertussis toxin, and be cross-linked by BMH. However, the propeller may be locally abnormal because mutations in repeats 2 and 3 diminish, but do not eliminate, the affinity for
. Blades 2 and 3 are the site of several residues that contact
, so decreased affinity because of local distortion in
these blades could be expected. In addition, mutation in repeat 3 exposes additional sites to digestion by trypsin. Nevertheless, the end
state for the mutants is close to normal.
Synthesis of mutant in COS-7 cells allows us to evaluate the
final state but does not allow us to analyze the effect of the mutation
on the folding process. Such analysis can be done with subunits
synthesized separately in vitro, then mixed, and assembled.
Folding and assembly are intimately linked for G
and G
. We have
shown previously that without G
, G
synthesized in vitro does not fold into a native structure (9, 11). At best, the
reticulocyte lysate is inefficient at folding and/or assembling
,
and only 20-30% of the wild-type
synthesized forms a dimer. In vitro dimerization of G
stops after about 30 min,
perhaps because an essential protein chaperone or cofactor is degraded or depleted. Adding fresh lysate increases the dimerization of both
wild-type and mutant protein, but we were not able to drive the
reaction to completion.
Mutation of the Asp in repeat 1 decreases the recovery of to
25% of wild type. Kinetic analysis shows that over the first 30 min of
folding and assembly, the rate of
formation by this mutant is
28% of wild type. Thus, the low recovery of H
1[D1]
dimers reflects the slowed rate of formation. The recovery of other
mutants varies from nearly undetectable for repeat 7 to nearly normal
for repeat 2. The dimerization rate of the mutants that were further
analyzed (H
1[D3], H
1[D4],
H
1[D5]) was also found to be low, and varied from one
to another, consistent with their specific yield of
dimers. The
slower rate of folding and assembly of the mutants may be due to the
flexibility introduced by glycine that increases the number of
potential conformations and so increases the time needed to find the
correct one. Alternatively, mutation may lower the affinity of the
protein for an essential chaperone. Whatever the mechanism, these
results show that mutations in each repeat do not have the same
consequences for folding and assembly, and they are not equivalent.
The comparison of in vivo and in vitro results indicates that the Asp mutants may be folding pathway mutants; they are able to achieve a normal or near normal structure, but the folding process is slowed. Therefore, they can be considered as conditional mutants whose phenotype is only evident under non-permissive conditions, such as a reticulocyte lysate, where elements important for folding are limiting. These mutants may provide an assay to identify factors and/or conditions that suppress or diminish their phenotype in a reticulocyte lysate. Identifying such factors might help understand the folding process of this WD repeat protein and perhaps of other members of the family.
Each of the Asp to Gly mutants in Sec13 were able to fold into a
compact globular structure (presumably a propeller) with the same
Stokes radius as the native protein. Unlike G, they are capable of
doing so even in vitro, consistent with our previous finding
(8, 11) that Sec13 has a less stringent requirement for chaperonins, or
can use chaperonins from very different sources (E. coli to
mammalian cells).
Although all the mutant Sec13 proteins had a normal Stokes radius, not all of them were resistant to trypsin. Because the 28 potential tryptic cleavage sites in Sec13 are randomly distributed, the cleavage assay is extremely sensitive and can detect even subtle conformational changes. We propose that the Asp to Gly mutation provides a certain freedom for the propeller to "breathe" and thus allows access of the enzyme to otherwise hidden sequences.
Comparison of the effect of Asp mutations in with the equivalent
mutations in Sec13 shows that the repeat positions that have the least
effect on the final structure are different for the two proteins
(repeat 2 in G
and repeats 1 and 6 in Sec13). All known propeller
proteins have a mechanism for closing the ring (reviewed in Ref. 6). In
G
, and presumably in all other WD proteins, the outermost
strand
of the last blade is provided by the amino-terminal variable region of
the first repeat, thereby bringing together the two ends of the
molecule to create the circular structure. Thus, one would expect that
the first and last repeats would be more sensitive than others to any
mutation. Our results prove that this assumption is wrong because, in
Sec13, the first and last repeats are precisely the ones where Asp
mutations have no detectable phenotype.
Our finding, both in G and in Sec13 that no mutation of the
conserved Asp entirely prevents folding, suggests that there is no
obligatory folding order of the repeats. Harrison and Durbin (7)
proposed that evolution favors multiple paths leading to the same final
folded state. One can easily imagine that formation of the D-containing
tight turn between strands b and c is one of the early folding steps
acting as a seed for each blade. So long as at least a majority of
blades can initiate folding under optimal conditions, nearly any folded
subset of blades will initiate the overall propeller. Nevertheless,
analysis of in vitro folding and assembly of G
suggests
that not all pathways are equally favorable because mutation of a
critical residue in one blade has a different effect from mutation in
another. We propose that different WD repeats initiate different
folding pathways with different rates or probabilities, which may
differ in different WD proteins.
Wall et al. (5) and Lambright et al. (3) pointed
out that the conserved Asp forms intra- and interblade hydrogen bonds in a triad with His in the GH motif, and a Ser/Thr in the second strand. Such a full triad occurs in only four (1, 3, 4, and 7) out of
seven repeats in G
, so triad hydrogen bonding is not absolutely
essential for proper folding or stability. However, our results suggest
that the presence or absence of the His that is hydrogen-bonded to Asp
can affect the final state of the molecule when Asp is mutated. In
G
, repeat 2 is the only one that lacks the His. It is also the only
one where mutation of the Asp does not slow folding in
vitro. Analysis of the double mutants is consistent with the
conclusion that mutation in this repeat is better tolerated because
H
1[D2-3] and H
1[D2-7] fold with
similar efficiency as the single mutants, H
1[D3] and
H
1[D7]. In contrast, all other double mutants tested
were poorly folded. In Sec13, the only repeat with no His is repeat 6, and indeed, the double mutant, H
1[D1-6], shows the
same phenotype as the single mutants, H
1[D1] and
H
1[D6]. We could not test any more double mutants
because all other single mutants were already susceptible to trypsin
cleavage. Our hypothesis that mutation in Asp is best tolerated if His
is also absent is supported by analysis of 918 unique repeats. Overall,
36% of repeats lack the His. However, in the set of repeats that lack
Asp (15% of all repeats analyzed), 71% also lack His. Therefore, in
the whole set, when one element of the triad is missing (Asp), another (His) is preferentially absent.
If, as our data show, no single Asp is essential for the folding of the
WD protein, what force has conserved them? It is possible that the
conserved Asp could be involved in some still unknown function common
to all WD proteins. However, we speculate that the answer might be
related to the observation that, in general, two Asp mutations are much
more deleterious than one for a WD protein. In fact, there are very few
existing WD proteins lacking more than one of the conserved Asp. Only 5 out of 140 WD proteins analyzed have two non-conservative
substitutions, suggesting that in those cases, one of the two repeats
without the Asp could be an especially permissive one, similar to
repeat 2 in G. A possible explanation for the striking conservation
of these residues could be that a random substitution of one Asp would
put the protein at risk, because a second substitution would most
likely have dramatic effects on its ability to fold and its stability.
Such proteins would then be at a selective disadvantage and over time would disappear.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM36259 (to E. J. N).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.
Recipient of a Fellowship from "Consejo Superior de
Investigaciones Cientificas" (Spain).
¶ Supported by Grant P41 LM05205-12 from the National Library of Medicine.
To whom correspondence should be addressed: Cardiovascular
Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA
02115. Tel.: 617-732-5866; Fax: 617-732-5132.
1
The abbreviations used are: FBS, fetal bovine
serum; PBS, phosphate-buffered saline; H1, hexahistidine-tagged
1; HA, hemagglutinin; BMH, 1,6-bismaleimidohexane; PAGE,
polyacrylamide gel electrophoresis; wt, wild type.
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