(Received for publication, September 26, 1995; and in revised form, December 22, 1995)
From the
The transcriptional activation domain of the herpesvirus protein VP16 resides in the carboxyl-terminal 78 amino acids (residues 413-490). Fluorescence analyses of this domain indicated that critical amino acids are solvent-exposed in highly mobile segments. To examine interactions between VP16 and components of the basal transcriptional machinery, we incorporated (at position 442 or 473 of VP16) tryptophan analogs that can be selectively excited in complexes with other Trp-containing proteins. TATA-box binding protein (TBP) (but not transcription factor B (TFIIB)) caused concentration-dependent changes in the steady-state anisotropy of VP16, from which equilibrium binding constants were calculated. Quenching of the fluorescence from either position (442 or 473) was significantly affected by TBP, whereas TFIIB affected quenching only at position 473. 7-aza-Trp residues at either position showed a emission spectral shift in the presence of TBP (but not TFIIB), indicating a change to a more hydrophobic environment. In anisotropy decay experiments, TBP reduced the segmental motion at either position; in contrast, TFIIB induced a slight change only at position 473. Our results support models of TBP as a target protein for transcriptional activators and suggest that ordered structure in the VP16 activation domain is induced upon interaction with target proteins.
The herpes simplex virus virion protein VP16 is a potent
transcriptional activator that specifically activates viral immediate
early gene expression(1, 2) . As a transcriptional
regulatory protein, it contains two functional domains. The
amino-terminal portion of the protein, in association with host
cellular proteins, binds to specific sequences upstream of the
immediate early gene core promoters(3, 4) . The
transcriptional enhancement activity resides in the carboxyl-terminal
78 amino acids(5, 6) . This domain can strongly
activate transcription in various systems when attached to the
DNA-binding domain of a heterologous protein(7) . The VP16
activation domain is rich in acidic residues and has been regarded as a
prototype acidic activation domain (AAD)()(8) .
Extensive mutational studies of this domain have identified aromatic
and hydrophobic amino acids critical for its activity, for example the
Phe at position 442(9, 10) . These studies have
further suggested that the VP16 AAD contains two independent
subdomains: the N-subdomain (residues 413-456) and the
C-subdomain (residues 453-490) (10, 11, 12) .
The activation mechanisms of eukaryotic transcriptional activators have been the focus of many studies(13, 14) . In addition to alleviating chromatin-mediated inhibition(15) , activators have been proposed to interact with components of the basal transcription apparatus to stimulate or stabilize the formation of the transcription initiation complex at the promoter. Biochemical approaches have identified several potential targets of activation domains, particularly for the AAD of VP16. TATA-box binding protein (TBP) was the first basal factor shown to directly bind to the VP16 AAD(16) . The specificity of this interaction was demonstrated by a correlation between binding of VP16 mutants to TBP and the transcription activities of these mutants(17) . Later, VP16 was shown to directly interact with another basal transcription factor, TFIIB(18) , although there is some discrepancy about the specificity of this interaction(11, 13, 19) . Recently, a specific interaction between VP16 and a subunit of transcription factor H has been reported(20) , as have interactions between VP16 and putative co-activator or adaptor proteins(11, 21) . Direct interactions between several of these target proteins and many other activation domains have also been shown(14) . Although the physical interactions have been demonstrated, their relevance and role in transcriptional activation are still largely unknown.
Despite abundant functional studies of
activation domains, little is known of their structures. No activation
domain structure has yet been solved by x-ray crystallographic analyses
or NMR. The limited biophysical studies of several AADs suggest that
isolated AADs are
unstructured(22, 23, 24, 25, 26) .
We recently performed fluorescence analyses employing chimeric proteins
comprising the GAL4 DNA-binding domain (residues 1-147) fused to
the VP16 activation domain(64) . Trp residues were substituted
for Phe at either position 442 or 473 of VP16, thus providing unique
fluorescence probes at two positions. Dynamic quenching, time-resolved
fluorescence decay, and time-dependent anisotropy decay studies showed
that the Trp residues at either position are solvent-exposed and highly
mobile. Our results, in agreement with CD and NMR studies of this
domain, reveal that the isolated VP16 AAD is poorly structured.
Noteworthy is that many of the biophysical studies suggest that under
certain conditions (low pH, hydrophobic solvent) these AADs can acquire
specific conformations such as helix and -sheet. These conditions
might mimic the in vivo conditions under which the AAD
interacts with its target proteins. The AADs, therefore, have been
hypothesized to adopt a specific conformation in the presence of their
target proteins. However, no structural characterizations of these AADs
have yet been carried out in the presence of their target proteins.
One major difficulty in studying protein-protein interactions by various biophysical means is that the signals from different proteins overlap and make the interpretation ambiguous. Recently, several groups reported that Trp analogs (5-hydroxytryptophan or 7-azatryptophan) can be successfully incorporated into proteins by using Trp auxotrophic Escherichia coli strains and supplementing the growth media with the relevant Trp analog(27, 28, 29) . The excitation spectra of these Trp analogs are shifted to longer wavelengths compared with Trp itself. Hence, fluorescence of proteins containing these Trp analogs can be selectively excited in the presence of other proteins containing natural Trp. Here we used this strategy to study the structural features of the VP16 AAD in the presence of two basal transcription factors, TBP and TFIIB. Our results indicate that the structure of the VP16 AAD becomes considerably constrained upon its interaction with these basal factors, particularly with TBP.
The steady-state fluorescence spectra were obtained with an SLM 8000 spectrofluorometer. The excitation wavelength was 309 nm. The emission spectra titration experiments of 7-aza-Trp-incorporated GAL4-VP16 were performed by recording the initial emission spectrum of the 4 µM 7-aza-Trp-incorporated GAL4-VP16 and then adding small aliquots of concentrated TBP or TFIIB solution and recording emission spectra until no further change could be detected. The same amounts of TBP or TFIIB were added to the buffer control, and these blank emission spectra were also recorded. Final emission spectra were corrected for blank control and for dilution.
Steady-state fluorescence anisotropy was measured using an L-format detection configuration. The excitation bandpass was 4 nm, and the emission bandpass was 8 nm. Excitation was at 309 nm, and emission was at 360 nm. Every data point was measured at least eight times. Data were fit to the equations describing formation of the 1:1 binary complex between GAL4-VP16 and TBP(36) ,
where r is the measured anisotropy when the fluorophore
is present in both the free form (5-OH-Trp incorporated GAL4-VP16) and
the bound form (complex with TBP), r and r
are the anisotropy of the free and bound
fluorophores, f
and f
refer
to the fraction of the total fluorophore that is present in the bound
and free forms, and I
and I
are the fluorescence intensities of the fluorophore in bound or
free forms, and,
where [V] is the total concentration of
the 5-OH-Trp-incorporated GAL4-VP16 used in the study and
[T]
is the concentration of the added TBP. K
is the dissociation constant for the association
between GAL4-VP16 and TBP. K
values were
determined using least-squares regression (IGOR, Wavemetrix, Lake
Oswego, OR).
Quenching experiments were performed at an excitation wavelength of 309 nm. Aliquots of 8 M acrylamide were added to 0.4 ml of 2 µM 5-OH-Trp-incorporated GAL4-VP16 (or mixtures of 2 µM 5-OH-Trp-incorporated GAL4-VP16 and 4 µM TBP or TFIIB) or 4 µM 7-aza-Trp-incorporated GAL4-VP16 (or mixtures of 4 µM 7-aza-Trp-incorporated GAL4-VP16 and 8 µM TBP or TFIIB) and to the appropriate solvent blank (0.4 ml of buffer or 4 or 8 µM TBP or TFIIB). The values of fluorescence emission intensity at 338 nm (for 5-OH-Trp-incorporated proteins) or at 396 nm (for 7-aza-Trp-incorporated proteins) were corrected for dilution and for blank. Quenching data were analyzed by the classic Stern-Volmer equation for dynamic quenching,
or by the single species dynamic-static quenching equation,
or by the two-species quenching equation (a rearrangement of Equation 11 in (37) ),
where F and F are the fluorescence
intensity in the absence and presence of quencher, respectively,
[Q] is the quencher concentration, and K
is the Stern-Volmer dynamic quenching constant. V is the
static quenching constant. f
is the fractional
contribution of the fluorophores that are accessible to the quencher,
and K
is the Stern-Volmer constant for the
accessible fraction. K
, V, f
, and K
values were
determined using least-squares regression (IGOR, Wavemetrix). The
bimolecular collisional quenching constant k
is
defined as follows,
where <> is the mean lifetime at zero quencher.
Time-resolved fluorescence was measured on a single photon counting
fluorometer(38) . Anisotropy decay curves were obtained by
alternatively recording the emission oriented parallel and
perpendicular to the plane of excitation. When the anisotropy decay
curves for the mixture of 2 µM 5-OH-Trp-incorporated
GAL4-VP16 with 4 µM TBP or TFIIB were recorded, those of
the appropriate blank (4 µM TBP or TFIIB) were also
recorded at the same time. Excitation wavelength was at 309 nm, and
emission wavelength was at 360 nm. Time per channel was 90 ps, and 512
channels were recorded. Data were corrected for blank control and
analyzed by the global analysis (39) or the ``sum and
difference'' method(40) . Rigorous confidence limits were
not measured for all parameters. Instead, several individual parameters
were explored by fixing their values to bracket those recovered.
Typical errors are approximately 10%, and typical
errors are approximately 5%. As discussed in (39) ,
values that are much larger than 6
become indistinguishable without further averaging.
Figure 1: Schematic representations of the various transactivators used in this study. All proteins contain the GAL4 DNA binding domain (amino acids 1-147) with tryptophan to valine substitution at position 36, designated as GV. All proteins also contain a 2- or 3-amino acid linker between the GAL4 domain and the VP16 domain. GV-5HW442 and GV-5HW473 are in-frame fusions of GV to the VP16 activation domain (amino acids 413-490) with the incorporation of 5-hydroxytryptophan at position 442 or 473, respectively. N-5HW442 contains a truncated VP16 activation domain (amino acids 411-456) with 5-hydroxytryptophan at position 442. GV-7AW442 and GV-7AW473 contain the full-length VP16 activation domain (amino acids 413-490) with the incorporation of 7-azatryptophan at position 442 or 473, respectively.
Figure 2: Spectroscopic properties of GAL4-VP16 fusion proteins bearing Trp analogs at position 442 or 473. A, peak-normalized absorbance spectra. B, peak-normalized excitation spectra. Emission was observed at 360 nm for GV-F442W and GV-5HW442, and at 380 nm for GV-7AW442. C, peak-normalized emission spectra with excitation at 310 nm. A-C, solid line, native GV-F442W; dotted line, GV-5HW442; dashed line, GV-7AW442.
The presence of 5-OH-Trp or 7-aza-Trp in the GAL4-VP16 proteins enables the fluorescence of the fusion proteins to be selectively excited at 310 nm in the presence of other Trp-containing proteins. Recombinant basal transcription factors TBP and TFIIB were purified from E. coli, and their transcriptional activities were confirmed using specifically depleted nuclear extracts (data not shown). As expected, these proteins were not efficiently excited using 310-nm light; the fluorescence observed for a 2-fold molar excess of TBP or TFIIB when excited at 310 nm amounted to less than 10% of the signal observed for GAL4-VP16 proteins bearing Trp analogs in the presence of a 2-fold molar excess of TBP or TFIIB.
Figure 3:
Effects of TBP and TFIIB on fluorescence
emission spectra of GV-7AW442 (panel A) and GV-7AW473 (panel B) at 310-nm excitation. The activator proteins were
present at 4 µM in these experiments. Solid line,
activator alone; dotted line, titration with 2 µM TBP; dashed line, titration with 4 µM TBP; dash-dotted line, titration with 8 µM TBP. The
ratios of emission intensity (F/F
) of GV-7AW442 and
GV-7AW473 in the presence of TBP (triangles) and TFIIB (squares) are shown in panels C and D,
respectively.
It should also be noted that the quantum efficiency of GV-7AW442 and GV-7AW473 increased modestly in the presence of TBP. The relative intensity around 370 nm region increased; however, the emission maximum is still at 396 nm. These fluorescence properties most closely match those of the model compound 7-azaindole in alcohol rather than those of 7-azaindole in aprotic solvents such as acetonitrile(41) . Hydroxyl groups in alcohols induce tautomerization of 7-azaindole, resulting in two populations of fluorescing molecules(29, 41) . The results of this study imply that the surroundings of both 7AW-442 and 7AW-473 become more hydrophobic; however, either the solvent is not totally excluded from these residues or there are nearby polar residues that hydrogen bond to the 7AW to cause tautomerization.
Figure 4: Stern-Volmer plots for the quenching of the fluorescence of GV-5HW442 (panel A) and GV-5HW473 (panel B) by acrylamide. 2 µM of the activators and 4 µM of TBP or TFIIB were used in these experiments. Closed circles, activator alone; triangles, in the presence of TBP; squares, in the presence of TFIIB. Data sets were compared with the various quenching models described under ``Experimental Procedures.'' The solid line represents the quenching model to which the data are best fit.
In the
presence of saturating amounts of TBP, the response of both GV-5HW442
and GV-5HW473 to acrylamide changed significantly. The Stern-Volmer
plots of both proteins were linear, and the data were best fit to the
purely dynamic quenching model, with a K of 8.2 M
and 6.3 M
,
respectively. In this case, the dynamic quenching rate constant was 2.6 M
ns
for GV-5HW442 and
2.2 M
ns
for GV-5HW473.
In the presence of a similar amount of TFIIB, the Stern-Volmer plots of
both proteins showed upward curvature, as observed for those of the
labeled fusion proteins alone. The analysis gave a K
of 5.7 M
and 3.9 M
and a static quenching constant V of 1.0 M
and 1.6 M
, respectively. The dynamic quenching rate
constant was 2.2 M
ns
for GV-5HW442 and 1.5 M
ns
for GV-5HW473. Although both TBP and TFIIB
altered the quenching effect of acrylamide for both probes, the nature
of the effect is very different in the two cases. The presence of TBP
eliminated the static quenching process, whereas it did not change the
dynamic quenching process significantly. In contrast, in the presence
of TFIIB both static and dynamic quenching remain, albeit somewhat
altered. Moreover, the addition of TBP to the truncated activator
N-5HW442 altered the quenching rate by acrylamide as it did to the
full-length AAD, whereas the addition of TFIIB had no effect on
accessibility (data not shown). Thus, if there is any interaction
between TFIIB and the activators, the effect of that interaction on the
structure of the VP16 AAD is apparently different from that seen with
TBP.
The activator proteins containing 7-aza-Trp were also tested in
acrylamide quenching assays (Fig. 5, A and B).
Stern-Volmer plots of acrylamide quenching of GV-7AW442 and GV-7AW473
were linear, yielding a K of 2.3 M
and 3.3 M
,
respectively (Table 2). Acrylamide was a less efficient quencher
for 7-aza-Trp than for Trp or 5-OH-Trp. The presence of TBP altered the
solvent accessibility of both residue 442 and 473. The downward curves
of the Stern-Volmer plots were best fit to a two-species model, with
approximately 40% of the probe molecules being inaccessible to
acrylamide (assumed K
of 0) and an accessible
fraction of approximately 60% having a K
of 4.0 M
or 7.0 M
for
GV-7AW442 or GV-7AW473, respectively. In contrast, the presence of
TFIIB with GV-7AW442 did not change the quenching mechanism, nor did it
change significantly K
. However, TFIIB did alter
the solvent accessibility of GV-7AW473. Its Stern-Volmer plot was
downward curved; in a two-species model, approximately 30% of the probe
was inaccessible, and the accessible fraction had a K
of 4.9 M
. In this case, TFIIB caused
a change similar to that seen with TBP.
Figure 5: Stern-Volmer plots for the quenching of the fluorescence of GV-7AW442 (panel A) and GV-7AW473 (panel B) by acrylamide. 4 µM of the activators and 8 µM of TBP or TFIIB were used in these experiments. Closed circles, activator alone; triangles, in the presence of TBP; squares, in the presence of TFIIB. Data sets were compared with the various quenching models described under ``Experimental Procedures.'' The solid line represents the quenching model to which the data are best fit.
Figure 6:
Steady-state anisotropy analysis of
GV-5HW442 (panel A), N-5HW442 (panel B), and
GV-5HW473 (panel C) in the presence of TBP or TFIIB. 2
µM of the activators were used in these experiments. Triangles, titration of the activator with TBP; squares, titration of the activator with TFIIB. The solid
line represents the best fit of the data to the equation
describing formation of a 1:1 binary complex of activator and TBP. The
calculated dissociation constants for GV-5HW442/TBP, N-5HW442/TBP, and
GV-5HW473/TBP interaction are 3.3 (± 1.7)
10
, 3.8 (± 1.4)
10
, and 2.8 (± 0.6)
10
M, respectively.
In contrast, the
addition of TFIIB to GV-5HW442 had little or no effect on anisotropy (Fig. 6A). We infer either that GV-5HW442 did not form
a complex with TFIIB, or that even if a complex formed, TFIIB did not
change the segmental motion of the fluorophore in the GV-5HW442.
Similar effects of TBP and TFIIB were observed for the truncated fusion
protein bearing 5-OH-Trp at position 442 of N-5HW442 (Fig. 6B). The calculated dissociation constant for the
interaction between TBP and N-5HW442 is 3.8 (± 1.4)
10
M. When the analog was incorporated at
position 473 of the full-length activation domain (GV-5HW473), the
anisotropy also increased rapidly as TBP was added to the system, and
the anisotropy reached a limiting value (Fig. 6C). The
calculated dissociation constant for this interaction is 2.6 (±
0.6)
10
M. Again, the addition of
TFIIB to the same protein caused no significant anisotropy change.
Figure 7: Time-resolved anisotropy decay curves of GV-5HW442 (panel A), N-5HW442 (panel B), and GV-5HW473 (panel C) in the absence or presence of TBP or TFIIB. 2 µM of the activators and 4 µM of TBP or TFIIB were used in these experiments. Smoothed curves of the raw data are shown. Solid line, activator alone; dotted line, in the presence of TBP; dash-dotted line, in the presence of TFIIB. A scaled lamp curve is given for reference (dashed line).
The anisotropy decay of these proteins was then measured in
the presence of a 2-fold molar excess of TBP or TFIIB. Under these
conditions, binding between GAL4-VP16 and TBP reached saturation as
indicated by steady state anisotropy titration experiments. In the
presence of TBP, the anisotropy decay of GV-5HW442 is greatly slowed (Fig. 7A). As for the activator fusion protein alone,
data were also best fitted to two decay components. However, the
contribution of the segmental motion () was
dramatically reduced from 75 to 40% of the total, and the slow
component dominated this decay process (Table 3). If one assumes
segmental motion can be reconciled with the ``wobble in
cone'' model (i.e. the localized motion of Trp is a
wobbling of its transition moment within a cone), the extent of this
motion can be described by the cone semiangle
magnitude(43, 44) . The cone semiangle for the
GV-5HW442 alone (52°) is larger than or comparable with those of
many known flexible polypeptides such as apocytochrome C with a
reported semiangle of 47°(42) . However, the presence of
TBP reduced the calculated cone semiangle to 35°. This result
indicates that the segmental motion around residue 442 in the VP16 AAD
is restricted in the presence of TBP. Moreover, the rotational
correlation time of the slow component (
) increased,
reflecting the change in molecular mass from the activation domain
alone to a complex containing GAL4-VP16 and TBP together. In contrast,
TFIIB did not cause any change in the anisotropy decay of GV-5HW442 (Fig. 7A). The lack of any effect on
suggests that TFIIB did not interact with the VP16 AAD or that if
any interaction does exist the mode of association has no effect on the
overall rotation of the AAD.
The anisotropy decay curves of the
chimeric protein containing the VP16 AAD N-subdomain in the presence of
TBP and TFIIB are shown in Fig. 7B. TBP formed a
complex with N-5HW442, evidenced by the increase in the rotational
correlation time () of the slow decay component (Table 3). TBP also reduced the amplitude (
) of
the segmental motion around residue 442, although the magnitude of the
effect is less than that seen for the full-length protein (GV-5HW442).
In contrast, TFIIB had much less of an effect on the anisotropy decay
of N-5HW442, consistent with the results seen for TFIIB with the full-
length protein labeled at position 442.
The anisotropy decay curves
of GAL4-VP16 labeled at position 473 of the full-length activation
domain (GV-5HW473) in the presence of TBP or TFIIB are shown in Fig. 7C. As observed with the probe at amino acid 442,
TBP greatly restricted the segmental motion around residue 473
(, Table 3). In addition, the apparent size of
the segment associated with this fluorophore at position 473 became
larger, as shown by the increment of the rotational correlation time of
the fast decay component (
). The rotational
correlation time of the slow component (
) also
increased, albeit not to the extent seen with the probe at position
442. In this case, the slow component may represent the
``freezing'' of a subdomain surrounding position 473 rather
than the size of the entire GAL4-VP16
TBP complex. This result
suggests that binding of TBP may have different effects on the
flexibility of the two subdomains of the VP16 AAD.
In this
experiment, the presence of TFIIB also affected the anisotropy decay,
reflected in an increase in the rotational correlation time of the slow
component (). The magnitude of this parameter was
smaller than that expected for the TFIIB
GV-5HW473 complex, and
thus probably represents only a subdomain of that complex. TFIIB also
moderately restricted the motion surrounding residue 473
(
). The extent of restriction was much smaller than
that caused by TBP; TBP reduced the cone semiangle of local motion from
47° to 31°, whereas TFIIB only reduced it to 43°.
Previous structural characterization of the AADs of VP16,
GAL4, GCN4, NF-B p65, and glucocorticoid receptor by CD and NMR
studies revealed that these domains were unstructured in aqueous
solution under neutral
pH(22, 23, 24, 25, 26) .
However, the AADs of VP16, NF-
B p65, and glucocorticoid receptor
all form an
-helix conformation in less polar
solvent(22, 23, 25, 26) . The AADs
of GAL4 and GCN4 form
-sheet in lower pH solution or in a
hydrophobic solvent(24) . Authors of these reports all
speculate that in the process of transcriptional activation, the AADs
adopt higher-order structure upon contacting their target molecules by
an ``induced fit'' mechanism. The present report provides
biophysical evidence to support that speculation, in that the local
structure surrounding key residues of the VP16 AAD was significantly
constrained upon interaction with TBP, and to a less extent, with
TFIIB. The induced conformations in transcription factors have been
previously shown only in the DNA binding basic region of leucine zipper
proteins and the arginine-rich RNA binding domain of human
immunodeficiency virus Tat protein (45, 46, 47) . The finding of the induced
ordered structure in the VP16 activation domain will likely lead to a
more refined analysis of the specific secondary and tertiary structures
induced by its target proteins.
In this study, Trp analogs with unique fluorescent properties were incorporated at key positions of the VP16 transcriptional activation domain. These spectrally enhanced proteins were used to study the interactions between this activation domain and the basal transcription factors TBP and TFIIB. In the absence of these factors, studies of the VP16 AAD containing 5-OH-Trp or 7-aza-Trp at positions 442 or 473 showed that both residues are solvent-exposed and are associated with highly mobile protein segments, consistent with our previous fluorescence analyses of the VP16 AAD containing natural Trp at these positions. The presence of TBP induced a significant change in the VP16 AAD, with a more ordered or constrained structure becoming apparent using fluorescent probes at either position. In contrast, effects of TFIIB interaction were observed only for probes at position 473 of the VP16 AAD, and those effects were weaker than those induced by TBP. Probes placed at positions 442 and 473 showed similar changes in the presence of basal transcription factor TBP. Probes at position 442 either in the full-length AAD or in the truncated subdomain also showed similar changes upon interaction with TBP.
The steady-state anisotropy
of GV-5HW442, N-5HW442, and GV-5HW473 increased substantially in the
presence of TBP. Dissociation constants were calculated from these
analyses. Dissociation constants between TBP and GV-5HW442 or N-5HW442
were both in the range of 3 10
M,
while that between TBP and GV-5HW473 was in the range of 3
10
M. The differences in these dissociation
constants may correspond to differences in transcriptional activities
as a result of the Phe
Trp mutations at positions 442 and 473.
The substitution mutant F442W retains 70% activity as a full-length
AAD, whereas the F473W mutation has a negligible effect on
activity(10) . An affinity capture method had previously
yielded an apparent dissociation constant of 2
10
M between the VP16 AAD and
S-labeled yeast
TBP(17) . The 10-fold difference in the results may be due to
inherent differences between spectroscopic and capture-type assays, or
to differences in the fusion protein constructs used in these
experiments.
Time-resolved anisotropy decay measurements demonstrate
that the mobility of protein segments surrounding positions 442 and 473
is markedly reduced in the presence of TBP ( Fig. 7and Table 3). When the VP16 AAD was labeled with 5-OH-Trp at either
position, the fraction of the anisotropy associated with fast decay
() was reduced by roughly 50% by binding to TBP, while
the fraction associated with slow decay (
) was
increased. Assuming that segmental motion can be correlated with the
fluorophore wobbling within a cone(43, 44) , the
calculated cone semiangle (Q) is reduced from approximately
50° to approximately 30°, representing a considerable
constraint on the segmental motion. Moreover, the increase in the
rotational correlation time for the slow decay component
(
) in the presence of TBP indicates that this
component is moving with a much greater mass. For the probe at position
442, this mass may approach that of the GAL4-VP16
TBP complex
altogether, whereas for the probe at position 473 the increase is less
dramatic and likely represents a somewhat smaller subdomain of the
complex. The rotational correlation time for the fast decay component
(
) also increased for the probe at position 473 (but
not for the probe at position 442), which may indicate that the fast
decay component results from a larger peptide segment surrounding 473
being induced by the binding of TBP. Curiously, a subtle difference can
be observed when the probe at position 442 is examined in the
full-length and truncated versions of the AAD. TBP apparently caused a
greater restriction of the segmental motion in the full-length AAD than
in the N-subdomain (compare calculated cone semiangles (
) for the
two AADs in the absence and presence of TBP). Nonetheless, the
rotational correlation times for the slow decay component
(
) of the truncated AAD increased, suggesting binding
between VP16 N-subdomain and TBP. Together, these results suggest that
the N-subdomain (surrounding Phe
) is the major targeting
site of TBP, but the C-subdomain still has some impact on this
TBP-activator interaction, either by providing a second, weaker binding
site or by modulating the TBP/N-subdomain interaction.
In contrast to the lack of effect on the N-subdomain, TFIIB did induce some changes in the fluorescence of the VP16 AAD with probes in the C-subdomain (position 473). TFIIB altered the quenching of GV-7AW473 by acrylamide (Fig. 5B), such that the quenching curves are best fit to a two-species model similar to that proposed for the effect of TBP. However, no shift in the emission spectrum of GV-7AW473 was observed in the presence of TFIIB (Fig. 3). TFIIB also partially protected both GV-5HW442 and GV-5HW473 from acrylamide quenching (Fig. 4), although the quenching mechanisms apparently retain both static and dynamic components, in contrast to the effect of TBP. This protection was C-subdomain-dependent, as it disappeared for N-5HW442 (data not shown). The differences in acrylamide quenching results for AAD proteins bearing 5-OH-Trp and 7-aza-Trp may be due to intrinsic differences in the quenching characteristics of these analogs. In sum, these results suggest that TFIIB may only sterically alter the accessibility of the quenching reagent without net changes in the polarity of the environment around residue 473, and thus no change in the emission spectrum of GV-7AW473 is induced.
TFIIB caused a modest
change in the anisotropy decay of GV-5HW473 (Fig. 7C and Table 3), although the effects were less striking than
those seen for TBP, and no noticeable effect was observed on the
anisotropy decay of GV-5HW442. In particular, the cone semiangle
() reduction caused by TFIIB is much smaller than that caused by
TBP. The magnitude of the effect on the rotational correlation times
for both the fast and slow decay components was approximately half that
observed with TBP, implying that the sizes of the domains responsible
for these components were not dramatically altered. The lack of any
significant change in steady-state anisotropy in the presence of TFIIB (Fig. 6C) might further suggest that the VP16-TFIIB
interaction is weak.
Taken together, these results indicate that the interaction of the VP16 AAD with TBP is very different from its interaction with TFIIB. TBP altered the fluorescence of probes at both 442 and 473, whereas TFIIB affected only probes at 473. Moreover, the magnitude of the effects induced by TBP was also consistently greater than those induced by TFIIB. While these results do not rule out the ability of TFIIB to interact with the VP16 AAD entirely, it is striking that few if any effects are observed on the properties of amino acids at or near positions critical to the transcriptional function of the VP16 AAD.
A second analogy is with
the binding of the basic pancreatic trypsin inhibitor to trypsin and to
trypsinogen, with dissociation constants of 10 and
10
M, respectively (49) . X-ray
crystallographic analysis shows that trypsinogen in complex with basic
pancreatic trypsin inhibitor complex acquires a trypsin-like
conformation (i.e. with a rigidly structured binding domain).
The reduced affinity of trypsinogen for basic pancreatic trypsin
inhibitor is a consequence of the energy required to order the binding
domain. Thermodynamic studies and structural comparisons have
demonstrated a large negative heat capacity change associated with
local or more extensive folding when a protein binds its ligand (or
another protein)(50) . In such systems, the binding energy from
the interaction creates part or all of the binding sites or even drives
folding beyond the interface.
Binding of a target protein to a flexible segment such as the VP16 AAD requires the reduction of its conformational entropy at the expense of association energy. Therefore, this kind of interaction, in which the flexible segment must be stabilized before it can provide optimal noncovalent interaction, is weaker than interaction with a rigid, stereochemically complementary surface. Nonetheless, an unstructured charged domain may have many advantages over a specific structured domain(51) . At neutral pH in aqueous solution, charge repulsion between the many ionized residues in these domains may inhibit formation of specific structure. These domains are therefore flexible and extend away from the proteins. The flexible and extended nature of these domains may increase the possibility of encountering the target proteins, and the charged amino acid side chains may provide a suitable force for promoting macromolecule association. Target proteins such as TBP and TFIIB present many surface-exposed basic amino acids that might serve to neutralize the acidic residues of the VP16 AAD and thereby help induce a specific conformation.
In addition to the charged or strongly polar amino acids commonly found in transcriptional activation domains, hydrophobic (and particularly aromatic) residues are often critical for the function of transcriptional activators(14) . Aromatic residues have been shown in several cases to provide the binding docking force for protein-ligand interactions (52, 53, 54) or to be directly involved in protein-protein interactions(55, 56, 57) . We speculate that the critical aromatic residues in the VP16 AAD participate directly in the binding of target proteins, providing some degree of binding stability and specificity.
The unusual potency of
VP16 as a transcriptional activator has been attributed to its ability
to bind to a number of target proteins in the transcriptional
apparatus(58) , which may allow VP16 to act during multiple
steps of preinitiation complex assembly(59) . The results of
this report do not contradict this multiple-target model. Although our
results demonstrate most clearly a specific interaction between TBP and
VP16 AAD, a weaker and more limited interaction with TFIIB was also
observed. Interestingly, a TBP mutant deficient in interacting with
TFIIB was shown to be deficient in GAL4-VP16 activated transcription (60) . This result suggests that in addition to interacting
with TBP directly, the AAD interacts with the
TBPTFIIB
promoter complex (61) . Thus, the weak
intrinsic interaction between VP16 AAD and TFIIB may be strengthened in
the presence of TBP.
Transcriptional activation is likely not to result from simple static interactions of activators with basal transcription factors but rather may involve the dynamic exchange of interactions among activation domains, basal factors, and coactivators. Recent studies show that distinct regions of the large subunit of RNA polymerase II share features in common with either acidic or proline-rich activators(62, 63) . The activation domains and the RNA polymerase II domains may compete for interaction with the same basal transcription factors or coactivators. If these interactions were to occur between rigid, stereochemically complementary protein surfaces, the binding might be so strong that exchange of such tight interactions would be difficult. In contrast, interaction of a target protein with flexible segments is weaker since association energy must be spent to compensate for the reduction of the conformational entropy. Thus, the transitions between ordered and disordered structures in activation domains (and their cognates in RNA polymerase II) may be a means to facilitate the dynamic interaction exchanges and hence to regulate the activation process.