From the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
Received for publication, March 5, 2003
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ABSTRACT |
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INTRODUCTION |
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Here we are concerned with the mechanism by which AA mediates the release
of F from the complex with AB. AB is a dimer and contains an
adenosine nucleotide binding pocket that can hold either ATP or ADP
(9). The binding of AB to
F is dependent on the presence of nucleotide in the pocket,
with ATP being more effective than ADP
(15). The complex is
asymmetric, having a stoichiometry of one molecule of
F per
dimer of AB (
F1·AB2)
(16). Previous work indicated
that AA reacts with the
F·AB·AB complex to
induce the release of
F from its antagonist
(12,
17). An attractive model for
the mechanism of this displacement reaction arose from the x-ray
crystallographic studies, which revealed a potential docking site for AA on
one of the two AB subunits
(18). According to this model,
AA would dock on the complex, making contact with one molecule of AB and
sterically displacing
F from the other molecule. Previous
work (13) had identified
Arg-20 of AB as a contact site for both
F and AA. A key
feature of the docking model is that Arg-20 on one AB subunit is in contact
with
F, whereas Arg-20 on the other subunit is exposed to
solvent and is free to make contact with AA. A second residue of importance in
the docking model is Glu-104 of AB, which is a contact site for AA but not for
F. Substitutions at Glu-104 impaired the capacity of free AB
to phosphorylate AA and prevent AA from causing the release of
F from the
F·AB·AB complex
(17). Here we describe the use
of heterodimeric mutant forms of the AB to investigate further the docking
model and other aspects of the reaction of AA with the
F·AB·AB complex.
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EXPERIMENTAL PROCEDURES |
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An expression plasmid for the purification of untagged AB in the experiment of Fig. 1 was made by amplifying the wild type gene from pDAG14 (13, 16) by PCR using oligonucleotides MH07 and MH08, which contained the restriction sites EcoRI and HindIII, respectively. The PCR product was digested with these enzymes and ligated to the expression plasmid pET-29a (Novagen), which had been similarly enzyme-treated, to create pMH13. Insertion into pET-29a joined spoIIAB to the coding sequence for an S-tag. pMH13 was introduced into E. coli strain BL21(DE3)/pLysS (Novagen) to create MHE31.
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Expression plasmids for the purification of His6-AB proteins were made by amplifying wild type and mutant forms of spoIIAB from pDAG14 (wild type spoIIAB gene), pMH84 (spoIIAB-E104K), pMH88 (spoIIAB-R20E), and pMH68 (spoIIAB-R105A) by PCR using oligonucleotides MH04 and MH11, which contained restriction sites for BamHI and XhoI, respectively. The PCR products were digested with these enzymes and ligated to the expression plasmid pRSETA (Invitrogen) that had been digested with these same enzymes to create pMH1 (His6-spoIIAB), pMH8 (His6-spoIIAB-E104K), pMH51 (His6-spoIIAB-R20E), and pMH54 (His6-spoIIAB-R105A). This fused the spoIIAB gene downstream and in-frame to six histidine codons in the vector. The plasmids were then transformed into BL21(DE3)/pLysS to create strains MHE08 (wild type), MHE53 (E104K mutant), MHE55 (R20E mutant), and MHE62 (R105A mutant).
The co-expression plasmid for producing the AB·ABR20E heterodimer was made by amplifying the wild type spoIIAB gene from pDAG14 using oligonucleotides MH04 and MH17, which contained BamHI and HindIII/EcoRI sites, respectively. (The oligonucleotide MH17 contains a HindIII site 4 bases away from the end of the EcoRI site, which was subsequently used for cloning the second mutant copy of spoIIAB gene between restriction sites HindIII and EcoRI.) The PCR product was digested with BamHI and EcoRI and ligated to the expression plasmid pGEX-2TK (Amersham Biosciences) that had also been treated with BamHI and EcoRI to create pMH21. This fused spoIIAB in-frame to the coding sequence for GST1 in the vector. By using pMH51 (the expression plasmid for producing His6-ABR20E) as a template, a DNA sequence containing the mutant spoIIAB, its ribosome-binding site, and the upstream histidine codons was amplified by PCR using oligonucleotides MH16 and MH11, which contained the restriction sites HindIII and EcoRI, respectively. The PCR product was digested with these enzymes and ligated to HindIII/EcoRI-digested pMH21 to create pMH46. pMH46 was transformed into BL21(DE3)/pLysS to create MHE21.
Construction of spoIIAC Expression PlasmidsStrain LDE7 used
for the production of F in the affinity chromatography
experiment of Fig. 3 was
described previously (9). The
spoIIAC expression plasmid used for the experiment of
Table I was constructed by
amplifying the spoIIAC gene from chromosomal DNA from B.
subtilis PY79 using the primers,
5'-atggatccatggatgtggaggttaag-3' and
5'-atgaattccatccgtatgatccat-3'. The PCR fragment was digested by
BamHI and EcoRI and ligated into the
BamHI/EcoRI-digested vector pGEX-2T (Amersham Biosciences)
to generate pMF14 in which the
F-coding sequence was fused
to the coding sequence for GST. pMF14 was then transformed into DH5
to
create MHE02.
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Protein PurificationE. coli strains used for the production
of AB and F were grown at 37 °C in 1 liter of LB
containing 75 µg/ml ampicillin and 25 µg/ml chloramphenicol until the
culture reached an A600 >0.4 (In the case of strains
used for producing AB heterodimer and
F, only ampicillin was
added to the culture.) T7 RNA polymerase synthesis was induced by the addition
of isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 1 mM. The cells were harvested 3 h later by
centrifugation. Cell pellets were suspended in the appropriate binding buffer
and disrupted by sonication. After sonication, the insoluble debris and the
unlysed cells were removed by centrifugation, and the supernatant fluid was
collected.
Four kinds of purification procedures were used for purifying AB and
F proteins. For purification of the untagged AB, cell
pellets from strain MHE31, which produces S-tagged AB, were suspended in
buffer A (20 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 1% Triton
X-100). The supernatant fluid was loaded onto S-protein-agarose (Novagen) for
1 h at 4 °C. After washing with 6 column volumes of buffer A, the
S-protein-agarose bound with S-tagged AB was suspended in buffer B (20
mM Tris-HCl, pH 8.4, 150 mM NaCl, 2.5 mM
CaCl2). 25 units of biotinylated thrombin were added to the agarose
mixture for up to 2 h at room temperature. Untagged AB was released from the
agarose during this cleavage reaction and stored in 15% glycerol at -80
°C.
For purification of the His6-AB (wild type and mutant proteins),
cell pellets from strains MHE08, MHE53, MHE55, and MHE62, which produce
His6-AB, His6-ABE104K,
His6-ABR20E, and His6-ABR105A
respectively, were suspended in buffer C (0.05 M Tris-HCl, pH 8,
0.5 NaCl, 5% (v/v) glycerol, 0.5 mM -mercaptoethanol) plus 5
mM imidazole. The supernatant fluid was loaded onto
Ni2+-NTA-agarose (Qiagen) for 1h at 4 °C. After washing with 6
column volumes of buffer C plus 20 mM imidazole, proteins bound to
the column were eluted with buffer C plus 200 mM imidazole. Elution
fractions containing the purified proteins were dialyzed into storage buffer
and stored in 15% glycerol at -80 °C.
Cell pellets from strain MHE21, which produced AB·ABR20E
heterodimer, were suspended in buffer C plus 5 mM imidazole. The
supernatant fluid was loaded onto Ni2+-NTA-agarose for 1 h at 4
°C. After washing with 6 column volumes with buffer C plus 20
mM imidazole, proteins bound to the column were then eluted with
buffer C plus 200 mM imidazole. Elution from
Ni2+-NTA-agarose was diluted and mixed directly with
glutathione-Sepharose 4B resin (Amersham Biosciences) with 0.5% (w/v) bovine
serum albumin (Sigma) for 1 h at 4 °C. The column was washed with 5 column
volumes of 1x PBS and eluted with buffer D (10 mM reduced
glutathione in 50 mM Tris-HCl, pH 8). The eluate was dialyzed into
buffer E (0.02 M Tris-HCl, pH 8, 0.15 M NaCl, 5% (v/v)
glycerol, 0.5 mM -mercaptoethanol, 5 mM
CaCl2) and treated with 50 units of thrombin. SpoIIAB heterodimers
were separated from the GST tag by remixing the fraction with
glutathione-Sepharose 4B resin for 1h at 4 °C. The flow-through from the
resin contained the pure SpoIIAB heterodimer. It was stored in 15%
glycerol.
Cell pellets from strain MHE02-produced GST-tagged F were
resuspended in 1x PBS. The supernatant fluid was loaded onto
glutathione-Sepharose 4B resin. The column underwent extensive washes with
1x PBS, and
F protein was released from the GST tag by
cleaving the column with 50 units of thrombin in 1x PBS.
Site-directed MutagenesisA mutant spoIIAB encoding ABR105A was created by site-directed mutagenesis using appropriately designed oligonucleotides MH85 and MH86, pDAG14, and the Quick-Change Site-directed mutagenesis kit (Stratagene).
Testing Subunit ExchangeEquimolar amounts (10 µM each) of AB and His6-AB were mixed and incubated at 4 °C for 3 h. The mixture was diluted in buffer C plus 5 mM imidazole from 50 to 500 µl. 25-µl resin (bed volume) was added to the 500-µl mixture, and the sample was rotated on a rotator at 4 °C for another hour. The resin was collected by centrifugation (4000 rpm), washed with 5 column volumes of buffer C plus 20 mM imidazole, and suspended in 25 µl of 2x SDS sample buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol). Eluates from the resin were analyzed by SDS-PAGE.
Surface Plasmon ResonanceKinetic and equilibrium constants of the protein-protein interactions were measured by surface plasmon resonance with a BIAcore 3000 instrument (Amersham Biosciences). To prepare the sensor chip, 80 µl of anti-His antibodies (concentration 50 µg/ml) diluted in 10 mM acetate, pH 4.5, were immobilized on the chip through the amino coupling procedure with HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.0005% surfactant P-20; Amersham Biosciences). The sensor surface was activated by injection of 1:1 mixture of N-hydroxysuccinimide/1-ethyl-3-(dimethylaminopropyl)carbodiimide for 8 min at 10 µl/min. Anti-His antibodies were then injected at a flow rate of 5 µl/min for 8 min. 1 M ethanolamine, pH 8.5, was injected at a flow rate of 10 µl/min for 8 min to block any unreacted esters. Unbound material near the chip surface was removed by injecting 10 mM HCl at a flow rate of 10 µl/min for 2 min.
A sample of solution containing His6-AB,
His6-ABR20E, or
His6-AB·ABR20E heterodimer diluted in HBS buffer
was applied to one flow cell of the CM5 sensor chip already coupled with
anti-His antibodies. The analyte F was dialyzed into binding
buffer (100 mM Tris-HCl, pH 7.4, 5 mM MgCl2,
0.5 mM dithiothreitol, 1 mM ATP) and injected over the
sensor surface at a flow rate of 30 µl/min for a total of 60 µl.
Concentrations of the analyte
F applied ranged from 20 to
200 nM. After the interaction, 50 µl of 20 mM HCl was
injected to regenerate the surface. Moreover, no specific interactions were
observed when analyte
F was injected over a flow cell with
immobilized anti-His antibody only. Apparent kinetic constants
(kon and koff) were obtained by use of
the BIAevaluation software (Amersham Biosciences).
Production of [35S]Methionine-labeled
FRadiolabeling of
F using
strain MHE5 was as described by Alper et al.
(15) with the following
changes. B1 vitamin was added to the culture (1 µg/ml). The
cells were grown in the presence of rifampicin for 2.5 h before labeling.
Unlabeled cells were harvested before the addition of
[35S]methionine, and 1 ml of the cultures was pelleted and lysed in
500 µl of buffer that contained 50 mM Tris-HCl, pH 8, 100
mM NaCl, 0.1 mM dithiothreitol, and 0.5% Triton
X-100.
Affinity ChromatographyMutant and wild type forms of AB
were mixed with Ni2+-NTA-agarose (30 µl of resin) for 1 h at
room temperature in buffer C. The resin was collected by centrifugation (4000
rpm). 100 µl of the radiolabeled F was added to the
resin, and the mixture (original volume of 150 µl) was diluted to a volume
of 500 µl with buffer C. The column support bed was formed with
5
µl of glass beads (200300 µM; Sigma, catalog number
G-1277), and the column was filled with buffer C. The column bed was formed by
adding the resin mixture and allowing it to settle while the column flowed by
gravity. The column was then washed 510 times with 5 column volumes of
buffer C. The column was eluted with 50 µl of 1% SDS, buffer C, 8
µM non-radioactive
F, or 8 µM AA
purified as described previously
(13).
Isoelectric FocusingRL2220 (thrC::spoIIQ-lacZ, erm spoIIE-1::kan) was transformed with chromosomal DNA from MHB10 and selected for spectinomycin resistance to create KC365. Cells were induced to sporulate by the resuspension method (25). At indicated times after the initiation of sporulation, 1-ml samples were harvested. For isoelectric focusing (IEF) cells were resuspended in lysis buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 mM MgCl2, 0.3 mg/ml phenylmethylsulfonyl fluoride, 0.5 mg/ml lysozyme, 0.1 mg/ml DNase I) and incubated at 37 °C for 10 min. Lysates were mixed 1:1 volume with 2x IEF sample buffer (8 M urea, 2.6% (v/v) ampholytes, pH 56 (Pharmalyte, Amersham Biosciences), 2% Triton X-100, 1% 2-mercaptoethanol, 0.04% bromphenol blue) and loaded onto a 5% polyacrylamide IEF slab gel containing 8 M urea and 2.6% (v/v) ampholytes, pH 56 (Pharmalyte, Amersham Biosciences). The gel was run at 200 V for 30 min followed by 300 V for 2.5 h with 10 mM phosphoric acid as the anolyte and 20 mM NaOH as the catholyte. Approximately equal numbers of cells were loaded for each sample as determined by A600 at the time of harvesting. The protein was electroblotted 20 V overnight (transfer buffer was 25 mM Tris, 193 mM glycine, 20% methanol) to Immobilon-P membrane (Millipore) and then incubated with affinity-purified polyclonal anti-SpoIIAA antibodies raised in rabbits against SpoIIAA purified as described previously (13). Donkey anti-rabbit antibodies labeled with iodine-125 (Amersham Biosciences) were used for detection on Biomax MS film (Eastman Kodak Co.).
Kinetic Measurements of Kinase ActivityPhosphorylation
reactions were carried out at 21 °C in 50-µl reaction volumes
containing 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10
mM MgCl2, 1 mM dithiothreitol, 1
mM ATP plus 5 µCi of [-32P]ATP (6000 Ci/mmol),
25 nM AB. The AA concentration was varied from 62.5 nM
to 2 µM. Reactions were stopped by the addition of 850 µl of
10% (v/v) trichloroacetic acid before the rate of phosphorylation reached a
maximum. The protein was precipitated by the addition of bovine serum albumin
to a final concentration of 1 mg/ml. The pellets were washed three times with
1 ml of 10% trichloroacetic acid and dissolved in 1 ml of Tris base. The final
sample was counted in liquid scintillation counter after adding 7 ml of
scintillation fluor (Opti-Fluor, Packard Instrument Co.). The time 0 blank was
subtracted from each value.
F-Directed
-Galactosidase
SynthesispMH68 containing the spoIIAB-R105A mutation was
linearized with restriction enzymes StuI and XcmI, and
introduced by double recombination into the spoIIA operon of
competent cells of MHB26 (thr::spoIIQ-lacZ::erm) by
transformation and selection for spectinomycin resistance, creating strain
MHB10. Cell pellets from MHB10 were collected at each time point from the
beginning of sporulation (time 0) in resuspension medium and treated with 10
µl of 20 mg/ml lysozyme at 30 °C for 15 min.
-Galactosidase
activity of the sample was determined according to Harwood and Cutting
(25).
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RESULTS |
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Purification of an AB HeterodimerArg-20 of AB was
identified previously as a probable contact site both for F
and AA in that a loss of side chain substitution (R20A) or a replacement with
a negatively charged side chain (R20E) impaired binding to either partner
protein in vitro and caused high levels of
F
activity in vivo
(13). The assignment of Arg-20
as a contact site for
F was confirmed by the recent
determination of the crystal structure of the
F·AB·AB complex
(18). The crystal structure
revealed that Arg-20 on one of the two AB subunits in the complex is in
contact with
F, whereas Arg-20 on the other subunit is
exposed to solvent and hence would potentially be available as a docking site
for AA. This interpretation predicts that heterodimeric AB in which one
subunit is wild type and the other harbors the R20E substitution would be
unimpaired in binding to
F. (We used R20E rather than R20A
because the effect of the former substitution is more severe than that of the
latter; see Refs. 13 and
17.) To create the
heterodimer, we co-expressed the genes for the mutant and wild type protein in
E. coli. The genes were arranged in tandem in a construct in which
their transcription was under the control of a promoter recognized by the
phage T7 RNA polymerase. The gene for the wild type protein in the construct
was preceded by the coding sequence for GST as well as the coding sequence for
the cleavage site for the protease thrombin, whereas the gene for the mutant
protein was preceded by six histidine codons. Thus, E. coli cells
harboring the construct were expected to produce GST-tagged AB and
His6-tagged ABR20E.
Expression of the construct was expected to generate three kinds of dimers: GST-AB homodimers, His6-ABR20E homodimers, and GST-AB·His6-ABR20E heterodimers. A lysate was prepared from cells of E. coli harboring the construct, and the following procedure was used to separate the heterodimers from the homodimers and other proteins in the lysates. First, we used Ni2+-NTA-agarose to purify His6-AB homodimers and GST-AB·His6-ABR20E heterodimers from the lysate. Lane 1 of Fig. 2 shows that both GST- and His6-tagged proteins had adhered to the column. Second, a glutathione-Sepharose (GST) resin was used to separate GST-AB·His6-ABR20E heterodimers from the His6-AB homodimers. The purified heterodimers (Fig. 2, lane 2) were treated with thrombin to remove the GST tag, resulting in purified AB·His6-ABR20E heterodimers. Finally, glutathione-Sepharose was used to remove the GST tag that had been released by thrombin treatment and residual GST-AB·His6-ABR20E heterodimers that had not undergone proteolytic cleavage from the AB·His6-ABR20E heterodimers. The purified AB·His6-ABR20E heterodimers were recovered in the flow-through fraction (Fig. 2, lane 3).
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By using similar procedures we also constructed and purified wild type homodimers of His6-tagged AB and mutant homodimers of His6-tagged ABR20E. For simplicity the two His6-tagged homodimers are henceforth referred to as AB·AB and ABR20E·ABR20E, and the His6-tagged heterodimer is called AB·ABR20E.
Kinetic and Equilibrium Constants for the Interaction of
F with SpoIIAB Homodimers and HeterodimersWe
used surface plasmon resonance to determine the equilibrium dissociation
constants for the interaction of
F with
His6-tagged AB·AB, AB·ABR20E, and
ABR20E·ABR20E. The purified homo- and
heterodimers (the ligands) were separately immobilized on a layer of dextran
that had been coupled with anti-His6 antibodies and attached to a
thin film of gold. Next, purified
F (the analyte) was
applied to the surface and allowed to bind with immobilized ligand during the
association phase of the analysis. After the analyte had been applied,
F was allowed to dissociate from the ligand during the
dissociation phase of the analysis. Association and dissociation of
F were detected optically. Software provided with the
instrument was used to calculate association rate constants from the
association phase of the interactions and dissociation rate constants from the
dissociation phase of the interactions. All interactions were monitored in the
presence of 1 mM ATP.
First we measured association and dissociation rate constants for the
interaction between AB·AB (wild type homodimer) and
F, from which we derived an equilibrium dissociation
constant (Kd) of 12 nM
(Table I). This value was
similar to that (14 nM) reported previously by Magnin et
al. (19). Second, we
investigated the interaction of
F with
AB·ABR20E. Importantly, we obtained a
Kd value that was only modestly higher (28
nM) than that observed with the wild type homodimer (see also
Table I). Finally, we attempted
to measure the interaction of
F with
ABR20E·ABR20E. The association rate constant
observed using the mutant homodimer was so low that an equilibrium
dissociation constant could not be derived, a finding that underscores the
importance of the Arg-20 side chain in the interaction of
F
with AB. In toto, these results indicate that complex formation
requires the presence of one and only one Arg-20 side chain, a finding
consistent with the idea that the
F-Arg-20 interaction
occurs on only one of the two subunits of the antisigma factor.
Rate constants obtained by surface plasmon resonance can differ
substantially from those obtained by other procedures and can be strongly
influenced by such factors as the flow rate of the analyte over the surface of
the sensor (20,
21). In this regard, we note
that the apparent rate constant for dissociation (koff)
for the F·AB·AB complex would appear to
indicate a relatively short half-life (
3 min). Yet other evidence from
affinity chromatography indicates that the complex is long lived (see Refs.
12 and
13; see below). With this
caveat in mind, the absolute values of the rate constants and equilibrium
dissociation constants presented in Table
I should be treated with caution. Nonetheless, we believe that we
are safe in the general conclusion that
F·AB·AB and
F·AB·ABR20E have similar
dissociation rate constants and much lower constants than that for
F·ABR20E·ABR20E.
A Complex of F with the AB Mutant
Heterodimer Is Resistant to AA-mediated DissociationPrevious work
indicated that Arg-20 on AB is responsible for contacting AA as well as
F. If, as our present results and those of Campbell et
al. (18) indicate,
F contacts Arg-20 on only one of the two subunits of the
antisigma factor, then Arg-20 on the other subunit might serve as a contact
site for AA during the AA-mediated dissociation of the
F·AB·AB complex. If so, then AA could cause
the release of
F from the
F·AB·AB complex by a simple displacement
mechanism in which the anti-antisigma factor docks on the subunit with a free
Arg-20 side chain. To investigate this hypothesis, we separately immobilized
AB·AB and AB·ABR20E on solid matrices. Next, we
applied radioactive
F to the matrices, thereby creating
immobilized complexes of
F·AB·AB and
F·AB· ABR20E. Finally, we measured
the release of radioactive
F from the complexes following
the application of buffer, buffer containing non-radioactive
F, and buffer containing purified AA.
Fig. 3a shows the
results of a representative experiment, and
Fig. 3b summarizes the
results of four independent experiments. As observed previously, only a small
proportion of the radioactive
F was released from the
F·AB·AB complex by buffer (5%; column
1 of Fig. 3b) or
buffer containing unlabeled
F (13%; column 2),
whereas a relatively high proportion (57%; column 4) was discharged
by the anti-antisigma factor. In contrast, AA was no more effective than
unlabeled
F or buffer alone in releasing radioactive
F from the
F·AB·ABR20E complex (7%; column
3). These results are consistent with a model in which AA docks on the
subunit with a free Arg-20 side chain to effect the release of
F from the other subunit.
The results of Fig.
3b also reinforce the view
(12,
13) that the
F·AB·AB complex is relatively long lived and
argue against an alternative model for the release of
F
based on the idea the complex is highly dynamic. In the dynamic model, the
complex undergoes rapid dissociation and re-association, with AA binding to
and trapping free AB·AB, thereby blocking the re-association of
AB·AB with
F. If the complex were dynamic, then
excess unlabeled
F should have been able to cause the
displacement of radioactive
F from the column of immobilized
F·AB·AB. Also, AA should have been able to
cause the release of
F from the
F·AB·ABR20E complex as effectively
as from the
F·AB·AB complex.
Efforts to Create a Catalytic Mutant Defective in the Phosphorylation
of AAAB is both an antisigma factor that binds to
F and a serine protein kinase that is capable of
phosphorylating AA (9,
10). Indeed, evidence
indicates that AA becomes phosphorylated when the anti-antisigma factor reacts
with the
F·AB·AB complex
(17). We therefore wondered
whether phosphorylation is required in the AA-mediated dissociation of
F·AB·AB or is simply a consequence of it. To
address this question, we sought to create a mutant of AB that was defective
in the kinase reaction but was unaltered in its ability to bind to
F and AA. Earlier work had shown that an amino acid
substitution at Glu-104 (E104K) impaired phosphorylation of AA, but kinetic
analysis revealed that the defect was due to impaired binding to the AA
substrate (Km) rather than impaired catalysis
(kcat)
(17).
The crystal structure of the F·AB·AB
complex reveals two candidates for amino acids that could be directly involved
in the kinase reaction. One candidate is Glu-46, which is in the catalytic
center of the kinase in a position from which it could promote the
nucleophilicity of the attacking water molecule in the ATPase reaction.
Accordingly, we built a loss of side chain substitution mutant in which Glu-46
was replaced with alanine. Unexpectedly, however, ABE46A proved to
be impaired in its ability to bind
F (data not shown). It is
known that the binding of AB to
F is dependent upon
adenosine nucleotide, and perhaps the Glu-46 side chain is needed to retain
ATP in the nucleotide binding pocket of the antisigma factor/kinase. In any
event, the E46A substitution is not simply impaired in catalysis and hence
could not be used to address the question of the role of phosphorylation in
the AA-mediated dissociation of the
F·AB·AB
complex.
A second candidate for a residue functioning in catalysis was Arg-105,
which as inferred from the crystal structure could be involved in stabilizing
the transition state of the phosphotransfer reaction as well as in contacting
Ser-59 in AA, which is immediately adjacent to the site of phosphorylation
(Ser-58). We built an R105A substitution mutant and found that it was
unimpaired in its ability to bind to F. Next, we carried out
a kinetic experiment to measure the rate of incorporation of
-32P into the substrate AA as catalyzed by
ABR105A and as compared with wild type AB. The Michaelis-Menten
curve of Fig. 4 shows that the
mutant enzyme was little altered in its catalytic activity
(kcat) but was markedly impaired in its
Km; we obtained a value of 557 nM for
the mutant enzyme as compared with 4.4 nM for the wild type kinase
(17). In this regard, the
R105A mutant resembles E104K, which as discussed above is also impaired in its
binding to AA. Evidently, the side chains of the adjacent residues Arg-105 and
Glu-104 are both needed for substrate binding but not for catalysis.
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ABR105A Is Defective in Phosphorylation of AA during SporulationNext, we carried out experiments monitoring phosphorylation of AA in vivo. Strains producing wild type AB or ABR105A were induced to sporulate, and samples were collected at 1.5 and 2 h after the onset of sporulation. Cell lysates were then prepared and subjected to IEF to separate unphosphorylated and phosphorylated (AA-P) forms of AA. AA and AA-P were visualized by immunoblotting with polyclonal anti-AA antibodies (Fig. 5). In a strain producing wild type AB, AA was phosphorylated with normal efficiency, as observed previously (13). Lanes 1 and 2 (1.5 and 2 h of sporulation, respectively) of Fig. 5 show both AA and AA-P, with the lower band representing AA-P. In striking contrast, we detected little or no AA-P in samples from cells producing ABR105A (lanes 3 and 4), suggesting that ABR105A is defective in phosphorylating AA. Note that the lower band present in lanes 3 and 4 is shifted slightly higher than the bands representing AA-P in the other lanes and represents one of two background bands that are also detected in a lysate of cells of a null mutant lacking AA (lane 9). To assess further the in vivo kinase activity of ABR105A, we monitored phosphorylation of AA in the absence of SpoIIE, the phosphatase responsible for dephosphorylating AA-P. As shown previously (6, 22) in cells lacking SpoIIE but producing wild type AB, AA was almost entirely in the phosphorylated form (lanes 5 and 6). Importantly, in cells lacking SpoIIE but producing ABR105A, a significant level of AA-P was observed, although the proportion of AA that was in the phosphorylated form was substantially lower than that observed with wild type AB (compare lanes 7 and 8 with lanes 5 and 6). We interpret these results to indicate that ABR105A is impaired but not completely blocked in kinase activity. Its residual kinase activity is masked by the action of SpoIIE but in the absence of the competing phosphatase kinase activity can be detected.
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ABE104K and ABR105A Have Opposite Effects on
F Activation in Vivo and on the Dissociation of the
F·AB·AB Complex in
VitroAs reported previously
(17) and confirmed here,
sporulating cells producing ABE104K are blocked in the induction of
a lacZ fusion to a gene under the control of
F.
Also, and again as confirmed here, the complex of
F with
ABE104K
(
F·ABE104K·ABE104K) is
known to be immune to attack by AA (9% release of radioactive
F; column 3,
Fig. 3d). We interpret
these results as indicating that
F does not become activated
in mutant cells simply because it is unable to escape from the
F·ABE104K·ABE104K
complex.
Remarkably, however, the R105A substitution had the opposite effect on
F-directed
-galactosidase synthesis in vivo
and on AA-mediated dissociation of the
F·AB·AB
complex in vitro. The results of
Fig. 6 show that in cells
producing ABR105A,
F was activated earlier and to
a much greater extent than in cells producing the wild type protein. Also, the
F·ABR105A·ABR105A
complex was susceptible to AA, efficiently releasing
F in
response to the anti-antisigma factor (59% release of radioactive
F, column 4 of
Fig. 3d).
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We interpret this result to indicate that AA-mediated release of
F is not dependent upon the kinase activity of AB. In
further support of this interpretation, we observed that approximately half of
the AA that eluted from the column with immobilized
F·AB·AB was phosphorylated, as detected by
isoelectric focusing, whereas almost all the AA that eluted from the column
with
F·ABR105A·ABR105A
was in the unphosphorylated form (data not shown). Approximately equal amounts
of
F were released from both columns but with little
phosphorylation of AA in the case of the complex with the mutant AB.
In summary, the E104K substitution impairs the capacity of AB to
phosphorylate AA and the ability of the
F·AB·AB complex to be dissociated by AA. The
R105A substitution, on the other hand, impairs the ability of AB to
phosphorylate AA but not the susceptibility of the
F·AB·AB complex to AA-mediated dissociation.
The simplest interpretation of these observations is that Arg-105 is only
needed in the kinase reaction and that AA-mediated dissociation of the
F·AB·AB complex does not require concomitant
phosphorylation of AA. In contrast, we presume that Glu-104 is an important
contact site between AB and AA both during the kinase reaction and during the
displacement of
F from AB.
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DISCUSSION |
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AB is both an anti-F factor and a protein kinase that is
responsible for phosphorylating and thereby inactivating AA
(9,
10,
11,
12). A point of uncertainty
has been whether AA becomes phosphorylated during the displacement reaction
and, if so, whether phosphorylation is required in order for AA to liberate
F from the complex. Campbell et al.
(18) demonstrated that a
non-phosphorylatable mutant of AA (AAS58A) in which the
phosphorylated residue, Ser-58, was replaced with alanine, was able to cause
the displacement of
F from the complex with AB. This finding
is consistent with the idea that displacement does not require concomitant
phosphorylation. We have been able to obtain complementary evidence on this
point through the creation of an amino acid substitution mutant of AB at
residue Arg-105 (R105A). The ABR105A mutant was defective in its
capacity to phosphorylate AA, as shown biochemically as well as in
vivo. Nevertheless, the complex of the mutant protein with
F
(
F·ABR105A·ABR105A) was
fully susceptible to undergoing dissociation in response to wild type AA.
Moreover, cells producing ABR105A exhibited abnormally high levels
of
F activity during sporulation, a finding consistent with
the idea that the mutant is defective in phosphorylating AA but not in
releasing
F. The crystallographic structure of the AA-AB
complex reveals that Arg-105 contacts the residue (Ser-59) that is immediately
adjacent to the side chain (that of Ser-58) that undergoes phosphorylation in
AA. Taken together, these findings are consistent with the idea that
ABR105A is involved in the kinase reaction but is not needed for
the interaction of AA with AB when
F is displaced from the
F·AB·AB complex. Thus, two complementary lines
of evidence, one based on a kinase-defective mutant of AB and the other on a
non-phosphorylatable mutant of the substrate AA, indicate that the
displacement reaction does not require concomitant phosphorylation of AA.
Nevertheless, it is entirely possible that AA does indeed become
phosphorylated in reacting with the complex, and previous evidence is
consistent with this idea. The conclusion we draw is that AA need not be
phosphorylated concomitantly in order for dissociation of the complex to take
place. Rather, it seems
likely2 that
displacement and phosphorylation represent successive steps in the reaction of
AA with the
F·AB·AB complex.
Finally, we comment on one additional feature of the interplay between AA
and AB. The antisigma factor and the anti-antisigma factor are mutually
antagonistic proteins. On the one hand, and as we have seen, AB that contains
ATP in its nucleotide binding pocket is capable of phosphorylating and thereby
inactivating AA. On the other hand, AA is capable of binding to an
ADP-containing form of AB (13,
15,
23). As a result of the kinase
reaction, AB is left with ADP in the nucleotide binding pocket. A fresh
molecule of unphosphorylated AA can bind tightly to the ADP-containing form of
AB to form a long lived complex. AB in the resulting AA·AB(ADP) complex
is inert both as a kinase and as an anti-F factor. Formation
of the AA·AB(ADP) complex is believed to contribute importantly to the
activation of
F by sequestering AB in a form in which it is
unable to phosphorylate AA or to inhibit
F
(15,
24). Recent work by Masuda
et al.3
reveals that AA undergoes a conformational distortion upon binding to
ATP-containing AB. The authors hypothesize that this energetically unfavorable
distortion does not occur when AA binds to the ADP-containing form of AB,
thereby explaining the high stability of the AA·AB(ADP) complex.
These findings also shed new light on the role of residue Glu-104 in AB. As
confirmed here and shown previously, the Glu-104 side chain is required for
AB(ATP)-mediated phosphorylation of AA and in the AA-mediated displacement of
F from the
F·AB(ATP)2
complex. It is not, however, required in order for AA to form the
AA·AB(ADP) complex. Masuda et
al.3 propose that
the favorable electrostatic interaction of Glu-104 with AA compensates for the
energetic cost of the conformation distortion required in order for AA to
interact with the ATP-containing form of AB. Because no such distortion is
expected to occur when AA interacts with the ADP-containing form of AB,
Glu-104 is dispensable in AA·AB(ADP) complex formation. The findings
underscore the intricacy of the interplay between the proteins that govern the
activation of cell-specific transcription.
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FOOTNOTES |
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Supported by predoctoral fellowship from National Science Foundation.
To whom correspondence should be addressed: Dept. of Molecular and Cellular
Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138. Tel.:
617-495-1774; Fax: 617-496-4642; E-mail:
losick{at}mcb.harvard.edu.
1 The abbreviations used are: GST, glutathione S-transferase; NTA,
nitrilotriacetic acid; IEF, isoelectric focusing; PBS, phosphate-buffered
saline;
2 L. Campbell, personal communication.
3 S. Masuda, K. S. Murakami, E. A. Campbell, C. A. Olson, and S. A. Darst,
submitted for publication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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