 |
INTRODUCTION |
Bacteriophage T4 gene 32 protein, first isolated and characterized
30 years ago (1), has been a model for single strand-specific nucleic
acid-binding proteins (2). 32 protein1 binds cooperatively
to single-stranded nucleic acids, a property essential to its roles in
DNA replication, recombination, and repair as well as to its ability to
regulate its own expression at the translational level. The protein
consists of three domains, originally defined and generated by limited
proteolysis experiments (2). Each of these domains is structurally and
functionally distinct. For example, removal of the protein N-terminal
(B) domain to form the *II product results in the complete loss of
cooperativity (3). The presence of the C-terminal domain in *II reduces
somewhat the intrinsic affinity of the protein for single-stranded
nucleic acid (3). Thus, binding cooperativity is clearly the result of
homotypic protein-protein interactions that occur when the protein
binds nucleic acid.
The core domain (*III) contains the intrinsic nucleic acid binding site
(4-7). In the crystal structure of this domain complexed to a
single-stranded oligonucleotide, the substrate is located within a
positively charged cleft (6). Within this cleft is the internal
(Lys/Arg)3(Ser/Thr)2 (LAST) motif, which
we had previously predicted to be involved directly in nucleic acid
binding (8, 9). Although the oligonucleotide is disordered within the
crystal, modeling of the substrate clearly showed the basic side chains of Lys (110), Arg (111), and Lys (112) in contact with the nucleic acid
phosphates and plausibly positioned the heterocyclic bases in contact
with hydrophobic pockets (6). A large number of biophysical experiments
indicates that both electrostatic and nonpolar interactions contribute
to the overall binding free energy (2).
The C-terminal (A) domain has a major role in gene 32 protein
interaction with other T4 proteins (10, 11), a property that is the
basis of viewing this protein as a "candidate organizing factor"
for protein-protein interactions (12). This domain has another very
interesting property: it modulates the ability of the protein to lower
the melting temperature of natural double-stranded DNA helices. On the
basis of its selective binding affinity for single strands, the protein
should possess significant helix-destabilizing activity. However, the
intact protein fails to lower the Tm of natural
dsDNA, suggesting that there is a kinetic block to protein-induced
melting (1, 13). Curiously, proteolytic excision of the A domain from
the C terminus of 32 protein yields a product (*I) that lowers the
Tm of natural dsDNA by
60 °C, consistent with
the removal of the kinetic block. *I promotes rapid renaturation of
single-stranded DNA at temperatures below Tm (14). Under certain conditions, full-length 32 protein will renature DNA, but
it does not lower Tm under any of the tested conditions (13). Thus, in 32 protein, it appears that the presence of
the C-terminal domain establishes the kinetic barrier to DNA helix
destabilization. The presence of this domain has a small negative
effect on intrinsic nucleic acid binding affinity (3).
Although to a first approximation the binding properties of each domain
are independent of the others, there are indications that there are
functional linkages among them. We have observed that the residues
within the N-terminal domain most essential for homotypic
protein-protein interaction, Lys (3)-Arg (4)-Lys (5)-Ser (6)-Thr (7),
are homologous to the aforementioned LAST residues within the core
domain, Lys (110)-Arg (111)-Lys (112)-Thr (113)-Ser (114). On the basis
of this and other observations, we devised a model for the cooperative
binding of 32 protein (8, 9). In the absence of bound nucleic acid, we
proposed that there is an internal interaction between core domain LAST
residues and an acidic surface located elsewhere on the protein. Upon
cooperatively binding single-stranded nucleic acid, these LAST residues
now interact with the acidic nucleic acid backbone, and a
conformational change is effected whereby the N-terminal B domain is
positioned to interact with the acidic surface of the adjacent 32 protein. Thus, these residues are proposed to interact with both an
acidic protein surface and the acidic nucleic acid surface; the acidic protein surface is capable of interaction with both N-terminal and core
domain LAST sequences. The model links the functionality of nearly
identical sequences within the N-terminal and core domains, and if the
acidic surface is associated with the C-terminal domain (see below),
then all three domains become functionally linked.
Additional evidence that the domains are linked to each other comes
from the nucleic acid binding properties of full-length 32 protein with
mutations within the N-terminal LAST sequence at positions 3 and 4 (15,
16). Were the domains fully independent, these mutations would be
expected to affect only the cooperativity parameter,
, a measure of
homotypic protein-protein interaction. However, several mutants,
e.g. Arg (4)
Glu, affect both
and
Kint, the affinity of the protein for an
isolated binding site on the nucleic acid. Since the nucleic
acid-interactive surface within 32 protein is clearly within the core
domain, the alteration in Kint suggests that
there was a change of some sort in this surface brought about by a
change in the homotypic protein-protein interaction.
In our ongoing effort to understand how the protein modulates its
various functionalities through its structural domains, we provide in
this report answers to the following questions. 1) Do the N- or
C-terminal domains have an effect on the conformation of the core
domain? 2) Is the helix-destabilizing activity of *I a consequence of
its nucleic acid binding cooperativity? 3) Is the helix-destabilizing
activity of *I a direct consequence of its stoichiometric binding to
ssDNA? 4) Can different forms of 32 protein coordinately associate with
ssDNA, and can the binding of one form potentiate the binding of another?
We find that the presence of the C-terminal domain decreases the
proteolytic susceptibility of residues within the core domain. However,
the quenching of the core domain tryptophan residue fluorescence by
iodide is unaltered by the presence or absence of the terminal domains.
These results further support the notion that the C domain modulates
accessibility of large substrates to the nucleic acid binding surface,
but the overall conformation of the core domain remains largely
independent of the flanking regions.
We have studied in detail the helix denaturation and renaturation
activities of the three truncated products of gene 32 protein. In
addition, we have evaluated the nucleic acid helix-destabilizing activities of gene 32 protein lacking its C terminus (*I) in the presence of full-length protein or core domain (*III, lacking both N
and C domains). Our results indicate that under conditions (0.05 M NaCl) where *III shows no measurable effect on
Tm, the addition of this adduct increases the amount
of DNA melting in the presence of a fixed amount of *I. Similar results
are observed for the effect of intact protein on *I-effected DNA
melting. In both cases, although the hyperchromic change is enhanced,
the actual melting temperature increases. In general, the enhancement of hyperchromicity and increase in Tm is monotonic
with increasing amounts of intact protein or *III. These results
reflect binding of full-length protein or core domain to
single-stranded regions created by the action of *I and may be
indicative of protein-protein interactions between *I and intact
protein or *III.
 |
EXPERIMENTAL PROCEDURES |
Cloning, Expression, and Purification of Proteins--
pYS6 (17)
and pYS55 (6), expression plasmids encoding the 301-residue whole
protein and core domain (*III, residues 22-253), respectively, were
digested with EcoRI and NcoI. After gel
purification, the EcoRI-NcoI fragment of pYS6,
which spans codons 1-81 of the structural gene, was ligated with the
NcoI-EcoRI fragment of pYS55, which spans codons
81 to 253. This produced plasmid pEKF1 (encoding *I, residues 1-253),
and conversely, the corresponding fragments of pYS6 and pYS55 were
ligated to produce plasmid pEKF2 (encoding *II, residues 22-301). The
scheme is shown in Fig. 1. Restriction analysis and sequencing of the plasmids confirmed the identity of the
clones.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Cloning scheme for pEKF1 and pEKF2, plasmids
encoding *I and *II, respectively. The parent plasmids, pYS6 and
pYS55, encode full-length protein and *III (core domain),
respectively.
|
|
The plasmids were transformed into the AR120 Escherichia
coli cell line. Expression was induced with 0.1 mg/ml nalidixic
acid when the cells reached an A595 of 0.8-0.9,
and the proteins were isolated on denatured DNA-cellulose as described
previously (7, 17, 18). The chromatographic properties of *I were
similar to that of whole protein; peak elution was at ~0.8
M NaCl. Likewise, both *II and *III eluted at
0.5
M NaCl. In all cases, the yield of purified protein was
>30 mg/liter of cell culture. SDS-polyacrylamide gel electrophoresis
indicated that the proteins were of >95% purity and had the expected
molecular weights. Protein concentrations were determined
spectrophotometrically, using
280M = 3.7 × 104 M
1
cm
1 (13). Note that all the Trp and Tyr
residues of 32 protein are located within the core domain.
Other Materials--
Calf thymus DNA (Sigma) and poly[d(A-T)]
(Amersham Pharmacia Biotech) were dissolved in the appropriate buffer,
stored at
20 °C, and used without further purification.
Endoproteinase Arg-C from mouse submaxillary gland (EC
3.4.21.35; type XX-S) was obtained from Sigma as a lyophilized
powder. Specific activities were typically 570 units/mg of protein (1 unit hydrolyzes 1.0 µmol of
N-p-tosyl-L-arginine methyl ester
min
1 at pH 8.0 and 25 °C). The enzyme was
dissolved in H2O and stored at -20 °C.
Proteolysis Experiments--
Proteolysis experiments utilized
endoproteinase Arg-C from mouse submaxillary gland and were conducted
at 37 °C with an enzyme:protein mass ratio of 1:10 (7) in 8 mM Tris-HCl, pH 8.1, 28 or 39 mM NaCl (as
indicated in the captions to Figs. 2 and 7 and Table III), 0.4 mM EDTA, 4% glycerol, 0.4 mM
-mercaptoethanol. Part of the NaCl concentration is contributed by
the salt content of the commercial enzyme preparation. After incubation
for the indicated period of time at 37 °C, the reaction was
terminated with SDS. Each reaction mixture or aliquot was applied to a
10-20% polyacrylamide Tris/Tricine SDS gel (Novex) or, for kinetic
experiments, to a standard 15% Tris/glycine SDS gel and then
electrophoresed. The gel was stained in 0.1% Coomassie Blue R in 40%
methanol, 10% acetic acid and destained in the same solvent. The 15%
Tris/glycine gel easily resolved *III from *I.
Fluorescence Experiments--
Iodide quenching experiments were
performed as described by Kelly and von Hippel (19). Fluorescence
measurements were performed at 25.0 °C using 10-mm × 10-mm
cells in a SPEX Fluoromax II spectrofluorimeter. Aliquots of a 1.00 M KI solution treated with 1 × 10
4 M
Na2S203 to prevent formation of
I3
(20), were added to 1000-µl
solutions containing 1.00 µM intact gene 32 protein or
core domain (*III) in 50 mM
Na2HP04, 1 mM Na2EDTA, 1 mM
-mercaptoethanol, pH 7.7. The results were analyzed
according to the Stern-Volmer equation,
Fo/F = 1 + KQ[Q], where Fo = emission of
unquenched protein, F = emission of quenched protein,
[Q] = concentration of quencher, and KQ is the
quenching constant. Plots of Fo/F were
subjected to linear regression analysis; the uncertainties in the
calculated values of KQ were obtained from the S.D.
of the slopes of these plots.
DNA Melting and Renaturation Monitored
Electrophoretically--
A 75-base pair HinfI restriction
fragment of pBR322 (36% G+C) from a 1-kilobase DNA ladder (Life
Technologies, Inc.) was isolated by electroelution into a dialysis bag
and labeled with [
-32P]ATP using calf intestinal
alkaline phosphatase and T4 polynucleotide kinase. The denatured form
of this fragment was prepared by heating the DNA to 95 °C followed
by rapid quenching. Reactions were performed in either 5 mM
Tris-HCl, pH 7.5, 0.5 mM EDTA (low salt buffer) or in 5 mM Tris-HCL, pH 7.5, 0.5 mM EDTA, 50 mM NaCl, and 10 mM MgCl2 (high salt
buffer) at 37 °C for 30 min. 1 pmol of DNA (residue) and 15 pmol of
protein (except where noted) were used in each reaction. Unless
otherwise noted, reactions were stopped by the addition of buffer to
create final concentrations of 0.33% SDS, 15% glycerol, and 0.15%
bromphenol blue. Electrophoresis was performed on 15 × 15-cm 10%
polyacrylamide gels (37.5:1 acrylamide/bisacrylamide) in 1 × TAE buffer (21).
Absorbance-Temperature Profiles--
Teflon-stoppered micro
quartz cuvettes containing 100 µl of test solutions were placed in a
Gilford 2400
2 spectrophotometer designed to raise the temperature at
a constant rate of 25 °C/h. Generally an absorbance-temperature
experiment uses a total of 4 cuvettes (a semimicro reference cuvette
containing
1 ml of buffer plus 3 micro cuvettes containing samples
of 100 µl). In formulating these solutions, the protein components
were mixed before the addition of nucleic acid so as to prevent any
order of addition problems. Temperature was continually monitored by means of a calibrated thermistor (Yellow Springs Instruments) inserted
through a narrow hole in the stopper of the reference cuvette;
absorbance was measured at 260 nm. Absorbance-temperature profiles were
graphically differentiated, and Tms were determined
two ways, as the inflection point of the transition and as the
temperature at which half the DNA was melted. Although the melting
profiles were not always symmetric, Tm values obtained by either method were within the stated uncertainties. The
reproducibility was about ±1 °C, except where otherwise noted.
 |
RESULTS |
The Presence of the C-terminal Domain Reduces the Susceptibility to
Proteolysis by Endoproteinase Arg-C--
We previously showed that
although the core domain of gene 32 protein was susceptible to the
action of mammalian endoproteinase Arg-C, the intact protein was
refractory to digestion (7, 9). To determine whether the presence of
the N- or the C-terminal domain is responsible for this protection, we
compared the endo Arg-C digestion behavior of all three truncated
products. As seen in Fig. 2, the
digestion pattern of *I is similar to that of *III, although the core
domain is somewhat more susceptible to the action of the enzyme. The
17- and 11-kDa bands produced by digestion of both *III and *I were
previously identified as being generated by cleavage at, respectively,
Arg-111 (corresponding to residues 112-253/4) and Arg-138
(corresponding to residues 139-253/4 (7)). Both these residues are
located within the nucleic acid binding cleft of core domain (6). In
contrast, virtually no low molecular weight products are seen when *II
is digested, where, as is the case with full-length protein, only small
amounts of a band corresponding to *III are observed (9). The
interacting oligonucleotide, p(dT)5, inhibits digestion at
these sites in both *III and *I but has no apparent effect on the
limited action of the protease on *II (Fig. 2). These results indicate
that the presence of the C-terminal domain either blocks access to the
arginines in the DNA binding cleft or alters the conformation of the
protein in the vicinity of these residues.

View larger version (104K):
[in this window]
[in a new window]
|
Fig. 2.
Comparative susceptibility to endoproteinase
Arg-C of gene 32 protein truncation products at 37 °C. Each
time point contained 0.22 nmol of the indicated protein and 1.1 units
of the protease in 12 µl of 8 mM Tris-HCl, pH 8.1, 28 mM NaCl, 0.4 mM EDTA, 4% glycerol, 0.4 mM -mercaptoethanol. When present, there are 6 nmol (p)
of p(dT)5/lane.
|
|
Quenching Experiments Indicate That the Local Conformation of
Tryptophan Residues within the Core Domain Is Unaffected by the
Presence of the N- and C-terminal Domains--
To further probe the
influence of the flanking regions on the conformation of core domain,
we have determined the effect of the collisional quencher, KI, on the
tryptophan fluorescence of intact protein and of core domain. All five
Trp residues are located within the core domain. Previous quenching
experiments suggest that binding of oligonucleotides exposes a
tryptophan side chain to the solvent environment (19).
Plots of the ratio of unquenched (Fo) to quenched
(F) fluorescence emission versus quencher
concentration (Stern-Volmer) plots are linear and are of identical
slope for both forms of the protein (Fig.
3; KQ = 5.03 ± 0.09 and 4.95 ± 0.10 for intact protein and core domain,
respectively). These results suggest that the iodide-induced quenching
is entirely due to a collisional mechanism, as noted previously for
intact protein by Kelly and von Hippel (19) and that the accessibility
of the Trp residues that are quenched is identical for both forms of the protein. Although one or two of the Trp residues in (folded) 32 protein are "dark" (2) and therefore do not serve as reporters for
KI quenching experiments, the identical slopes of the Stern-Volmer plots strongly indicates that the local conformation of the remaining tryptophans are equivalent. These results also suggest that the overall
conformation of the core domain is essentially independent of the
flanking N- and C-terminal domains, a conclusion consistent with a wide
body of data (2). Thus, the effect of the C domain on endoproteinase
Arg-C digestion is likely due to a reduction in accessibility of
Arg-110 and Arg-138 to the active site of the protease.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Stern-Volmer plots of full-length gene 32 protein (panel A) and core domain (panel
B) at 25.0 °C. In both cases, [protein] = 1.00 µM in 50 mM Na2HP04,
1 mM Na2EDTA, 1 mM
-mercaptoethanol, pH 7.7.
|
|
Intact Gene 32 Protein and Its Three Truncated Forms Differ in
Their DNA Helix-destabilizing and -renaturing Activities--
As we
noted in the Introduction, whole gene 32 protein is unable to lower the
Tm of long chain double-stranded DNA, and removal of
the C-terminal domain removes this inhibition. Initially, we wished to
assess and compare both the nucleic acid helix-destabilizing and
renaturing activities of intact and truncated gene 32 protein. To do
this, we incubated a 32P-labeled 75-base pair restriction
fragment, either double-stranded or heat-denatured, with each protein
for 30 min at 37 °C in either a very low ionic strength buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA) or in the
same buffer with 50 mM NaCl and 10 mM
MgCl2. The amount of protein used (15 pmol) was sufficient
to fully saturate ssDNA under conditions of high affinity. The two
ionic conditions were chosen so that in the absence of protein, the
Tm of DNA is above 37 °C in both buffers.
Likewise, based on the literature (14) and preliminary melting
experiments with calf thymus DNA, we predicted that in the presence of
*I, the Tm is below 37 °C at low salt
concentrations and above this temperature at high salt concentrations.
The results are shown in Fig. 4.

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 4.
DNA helix-destabilizing and renaturing
activities of full-length gene 32 protein and of truncated forms
lacking the C domain (*I) and the N domain (*II). A
32P-end-labeled 75-base pair DNA fragment (1 pmol),
double-stranded or heat-denatured, was incubated with protein (15 pmol,
except where noted) at 37 °C for 30 min in either low salt buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA) or high salt
buffer (5 mM Tris-HCL, pH 7.5, 0.5 mM EDTA, 50 mM NaCl, 10 mM MgCl2). Panel
A, in odd-numbered lanes, reactions were begun with
single-stranded DNA; in even-numbered lanes, double-stranded
DNA was initially present. The proteins and buffers were as follows.
Lanes 1 and 2, intact gene 32 protein, high salt
buffer. Lanes 3 and 4, *II, low salt buffer.
Lanes 5 and 6, *II, high salt buffer. Lanes
7 and 8, no protein present. Panel B, DNA as
in panel A. Lanes 1 and 2, *I, low
salt buffer. Lanes 3 and 4, high salt buffer.
Lanes 5 and 6, no protein present. Panel
C, in all lanes, DNA was initially double-stranded and
was incubated in low salt buffer with the following pmol levels of *I.
Lanes 1-6, 11, 5.5, 2.8, 1.4, 0.7, 0.35, respectively. This
corresponds to [*I]/[DNA]p = 0.16, 0.08, 0.04, 0.02, 0.01, 0.005.
|
|
Intact 32 protein is known to have no effect on the melting temperature
of natural double-stranded DNA (13). As shown in Fig. 4, panel
A, *II also failed to lower the Tm, since at
low ionic conditions it had no effect on either ss- or dsDNA. At high
salt conditions, both intact 32 protein and *II renatured single-stranded DNA to varying degrees (panel A). *I,
however, produced dramatic results under both low and high ionic
conditions. ssDNA was completely renatured at high salt, and dsDNA was
completely denatured at low salt conditions (panel B). Thus,
in the low ionic strength buffer, conditions where the temperature is
above the Tm of DNA in the presence of *I, this
protein stabilizes single-stranded DNA. When incubated in the higher
ionic strength buffer with either ssDNA or dsDNA, *I stabilizes dsDNA.
Thus, under conditions where intact protein or *II fail to lower
Tm or under conditions where these two proteins only
partially renature DNA, *I has the property of efficiently bringing DNA
to its equilibrium state. The helix-destabilizing activity is largely
lost when *I is pre-incubated with proteinase K (not shown). The
activity is clearly protein concentration-dependent, since
decreasing amounts of *I are less effective in denaturing dsDNA under
low salt conditions (panel C). Denaturing activity
correlates with site size; with a site size of about 7 (2, 22),
saturation of ssDNA should occur at a [*I]:[DNA]p = 0.14.
The C-terminal Domain Alone Is Responsible for the Kinetic Barrier
to DNA Helix Destabilization--
To localize the barrier to helix
destabilization, a spectrophotometric examination of DNA melting
activities of the truncated forms of DNA was undertaken. Under a
variety of conditions summarized in Table
I, *I displayed helix-destabilizing
activity with calf thymus DNA. *III was observed to lower
Tm only at very low salt concentrations,
4
mM NaCl (Table I); only partial melting was observed. No
measurable effect on Tm was observed at 50 mM NaCl. Even at very low NaCl concentrations (4.4 mM), no DNA melting was observed with *II up to the point
where the protein denatures (50 to 55 °C). Note that the affinities
of *II and *III for single-stranded DNA are similar, as are the
affinities of intact protein and *I for ssDNA (3, 22). Thus, analogous to intact protein, the likely reason for the failure of *II to lower
Tm is the presence of the C-terminal domain.
View this table:
[in this window]
[in a new window]
|
Table I
Melting of calf thymus DNA in the presence of truncated forms of gene
32 protein
[DNA]p = 30 µM (p) in a buffer containing 7.3 mM Tris-HCl, pH 8.1, 0.37 mM EDTA, 0.37 mM -mercaptoethanol, 3.7% (v/v) glycerol.
|
|
Intact Gene 32 Protein, Core Domain, and *II Alter the DNA
Helix-destabilizing Properties of the *I-truncated Protein: Evidence
for Coordinated Function--
Although neither intact protein nor *II
lowers the Tm of natural dsDNA and *III only
partially melts DNA, we have observed that all three proteins modulate
the melting activity of *I. Both Tm and the extent
of melting (hyperchromicity) are affected.
Typical profiles of *I-effected melting of calf thymus DNA with the
additional presence of intact protein or *III are shown in Fig.
5. At 50 mM NaCl, *I
decreased the melting temperature of calf thymus DNA from 78 to
23 °C. Under these conditions, none of the other forms of 32 protein
has any measurable effect on Tm. However, in the
presence of intact protein, *III, or *II, the *I-effected melting
profile yielded higher Tm values and increased the
hyperchromic change seen upon melting (Table
II). Typical melting profiles of calf
thymus DNA with *I and increasing amounts of *III or intact protein are
shown respectively in Fig. 5, A and B. With
[*I] held constant at 4.4 µM, the increase in
Tm and hyperchromicity was monotonic with increasing concentrations of the other protein up to a limiting value (Table II).
Similar results were observed with other concentrations of *I (data not
shown).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of core domain (*III, panel
A) and intact protein (panel B) on
*I-effected melting of calf thymus DNA. A, [*I] = 4.4 µM and [DNA] = 30 µM (p) in 50 mM NaCl, 7.3 mM Tris-HCl, pH 8.1, 0.37 mM EDTA, 0.37 mM -mercaptoethanol, and 3.7%
glycerol. , no *III; , 2.2 µM *III; , 3.3 µM *III. B, [*I] = 2.2 µM and
[CT DNA] = 30 µM (p) in 50 mM NaCl, 5.8 mM Tris-HCl, pH 8.1, 0.3 mM EDTA, 0.3 mM -mercaptoethanol, and 3.0% glycerol. , no 32 protein; , 2.2 µM 32 protein; , 4.4 µM 32 protein.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Effect of increasing amounts of *III, intact 32 protein, or *II on
*I-effected calf thymus DNA Tm depression
|
|
A comparison of the data in Fig. 5B suggests that intact 32 protein is somewhat more efficient than *III in raising the melting temperature and hyperchromicity. For example, with 4.4 µM
*I, the Tm was raised to 30 °C in the presence of
2.2 µM whole 32 protein, whereas 4.4 µM
*III had to be used to reach this point. Although high levels (8.8 µM) of *I effect essentially complete melting of the DNA
(A260 = 0.079 or 40% of the initial DNA
absorbance), this was not achieved with 4.4 µM *I and equal or
greater concentrations of intact protein or *III.
Although whole 32 protein on its own cannot lower the
Tm of natural double-stranded DNA, it can
destabilize the double-helix of poly[d(A-T)]. Analogous to the effect
of *III on *I-effected calf thymus Tm depression,
increasing amounts of the core domain brought about an increase in
poly[d(A-T)] Tm and hyperchromicity when added to
a fixed level of 32 protein and poly[d(A-T)] under conditions where
*III alone does not melt the DNA (Fig.
6).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of increasing amounts of *III on 32 protein-effected poly[d(A-T)] Tm and
hyperchromicity. [32 protein] = 3.3 µM and
[poly[d(A-T)]] = 31 µM (p) in 0.15 M
NaCl, 6.1 mM Tris-HCl, pH 8.1, 0.31 mM EDTA,
0.31 mM -mercaptoethanol, and 3.1% glycerol.
A, Tm. B,
hyperchromicity.
|
|
Proteolysis Experiments Suggest That the Affinity of *III for
Single-stranded DNA Is Increased in the Presence of
*I--
Endoproteinase Arg-C digestion experiments were performed to
examine if *III really bound to calf thymus DNA during the *I-effected melting process. Since the protein concentrations required in proteolysis experiments are much higher than those in melting experiments, the concentration of double-stranded CT DNA was raised accordingly to maintain an excess of DNA to *I, thus assuring there was
enough DNA available for *III binding. The Tm depression data at 50 mM NaCl showed that the maximum
Tm was around 33 °C. The ionic strength used in
these proteolysis experiments was lower (39 mM
Na+; Tm
28 °C), so that after the
30-min incubation at 37 °C the melting process would have been complete.
As shown in Table III and Fig.
7, in the absence of DNA the presence of
*I significantly reduces the digestion of *III. There is a general
inhibitory effect of double-stranded DNA on the digestion of *III, and
at a [*I]:[*III] ratio of 1.5:1, the digestion of *III was further
slowed down. Under these conditions, the digestion of *I was almost
totally inhibited. These results suggest that *I promoted the binding
to DNA of some of the core domain molecules, resulting in their further
protection against proteolysis. Experiments using the same buffer
conditions but with a 1:1 ratio of *I and *III also showed very strong
protection by DNA of *I but almost no protection of *III. These results
are consistent with the binding of *III to single-stranded CT DNA when
*I was present. The pattern of products generated by endo Arg-C
digestion as seen on the SDS gel was similar to those observed in
proteolysis experiments with oligonucleotides (7), indicating that the
interactions between protein and nucleic acid were probably the same in
both cases.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of double-stranded calf thymus DNA on
the endoproteinase Arg-C digestion kinetics of *III in the presence of
*I at 37 °C. The digestion mixtures contained 15 µM *I, 10 µM *III, 108 µM
(residues) double-stranded calf thymus DNA (if present), 0.05 mg/ml
(0.029 units/µl) endoproteinase Arg-C, 39 mM NaCl, 18 mM Tris-HCl, pH 8.1, 0.9 mM EDTA, 0.9 mM -mercaptoethanol, and 9% glycerol. Mixtures were
incubated for 30 min before the addition of the enzyme. Each time point
represents 3 µl of a mixture. , DNA present; , no DNA. Samples
were electrophoresed on 15% polyacrylamide Tris/glycine SDS gels, and
the relative amount of protein in each band was quantitated using a
Molecular Dynamics laser densitometer.
|
|
 |
DISCUSSION |
In this paper, we have further explored the effects of the C and N
domains on the nucleic acid-interactive properties of gene 32 protein.
The susceptibility of *III and *I to the action of mammalian
endoproteinase Arg-C indicates that the presence of the C domain
inhibits cleavage at Arg-111 and Arg-138, both located within the
nucleic acid binding cleft of core domain (6). Residue 111, which is
protected to a greater degree, is located within the internal LAST
motif (7). Likewise, the protective effect against digestion by a
binding oligonucleotide, p(dT)5, is observed for both *III
and *I. Thus, the presence of the N domain does not bring about any
changes in the accessibility of the nucleic acid binding site to the
action of the enzyme.
On the other hand, when the C domain is part of the polypeptide chain,
there is a major effect on proteolysis. We previously demonstrated that
the core domain within whole protein is refractory to digestion by endo
Arg-C (9). We have now observed the same result with *II. Thus, in this
truncated form as well as in the full-length protein, the C domain
clearly protects the core against the action of this protease.
To further probe the effect of the C-terminal domain on the core, we
conducted tryptophan quenching experiments with iodide ion. All the
tryptophan residues are located within the core domain, and one,
residue 144, is located close to the ssDNA binding groove (6). In this
study, we have obtained identical Stern-Volmer quenching plots for
full-length protein and core domain. Thus, the local conformation of
the reporting tryptophan residues is unaffected by the C domain.
Conceivably, there could be a difference in the environment of a dark
tryptophan (where the fluorescence is completely quenched upon protein folding).
The C-terminal third of gene 32 protein (residues 201 through 301),
corresponding to 50 residues of core domain and the (proteolytically defined) C-terminal domain is very acidic and is a potential mimic of
single-stranded DNA. In this regard, Gold and co-workers (10) demonstrate that this portion of gene 32 protein is particularly immunogenic, and they isolated several monoclonal antibodies with epitopes located within the C-terminal 100 amino acids. These antibodies strongly cross-reacted with single-stranded DNA.
Conceivably, the C-terminal third of the protein or portions of it
could mimic ssDNA and interact with core domain at the ssDNA binding
groove. The binding cleft can easily accommodate a polypeptide chain. This putative interaction is consistent with the protection against proteolysis at Arg-111 and Arg-138 seen in full-length protein and *II
as well as the slightly greater intrinsic binding affinity (Kint, for isolated binding sites) of *I and
*III relative to *II and intact protein (2, 3, 22).
As we noted in the Introduction, we have proposed that the N-terminal
and core domain LAST sequences could alternate binding the same acidic
surface, corresponding, respectively, to intermolecular and
intramolecular protein-protein interactions (8). The N-terminal LAST
sequence of a cooperatively bound gene 32 protein monomer would be
bound to the acidic surface of the adjacent DNA-bound monomer. When not
bound to nucleic acid, the core domain LAST sequence was envisaged to
be interacting internally with the acidic surface, i.e. of
the same molecule. The C-terminal third of the protein has a
theoretical pI of 3.9; the pI of the N-terminal two-thirds is 8.7. Thus, there is a strong likelihood that this acidic surface is located
within the C-terminal portion of the protein. Within the crystal
structure of the core domain (6), residues 201-239 constitute an
external flap that could swing into the ssDNA binding groove. The
remainder of the C domain, in intact protein, might also be involved in
such a conformational change. We note that Kowalczykowski et
al. (23) proposes the existence of an acidic flap that masks the
basic residues of the nucleic acid binding site but would be displaced
upon cooperatively binding single-stranded nucleic acid. A consequence
of our model is that since the acidic surface is envisioned to compete
with ssDNA for the DNA binding site, intermolecular protein-protein interaction, hence cooperativity, is not independent of intrinsic protein-nucleic acid binding. As we mentioned in the Introduction, Villemain and Giedroc (15, 16) show that certain mutations within the
N-terminal LAST sequence affected both the cooperativity parameter,
, and the intrinsic binding affinity for single-stranded DNA,
Kint. Our model provides an explanation for
these results.
The melting and renaturation studies presented herein further extend
our understanding of the roles of the domains of the protein in these
processes. The extent of the helix-destabilizing activity of *I is a
function of the degree of its binding saturation of single-stranded
DNA. Cooperative binding is not a prerequisite for the helix
destabilization of natural dsDNA, since core domain, which binds
single-stranded nucleic acids noncooperatively, brings about the
lowering of the Tm of natural double helices, although to a lesser degree than does *I. The extent of
Tm depression is a function of the relative
affinities of the protein for single-stranded versus dsDNA,
and largely due to the absence of cooperativity, the
Kassoc (Kint ×
) of
core for single-stranded nucleic acids is approximately 3 orders of
magnitude below that of *I (22). This assumes there is no major
difference in the weak affinity for dsDNA (intact protein binds
noncooperatively to dsDNA; Kassoc
104 M
1 in 0.05 M NaCl (13)). The C domain is the sole determining factor
in the ability to lower Tm; neither full-length protein nor *II destabilize natural dsDNA helices. If the C-terminal third of gene 32 protein interacts with the DNA binding site in the
absence of DNA, then it must be displaced to bind nucleic acid. Perhaps
the absence of the kinetic block to helix destabilization in *I and
*III reflects a more facile displacement from the ssDNA binding groove
of residues 201-253 than of residues 201-301 in *II and intact protein.
Our results suggest that this inhibitory activity can be mediated by
protein-protein interactions. In experiments where there was enough *I
to bring about the melting of only a portion of calf thymus DNA, the
addition of increasing amounts of either full-length or *II or *III
protein monotonically increased the fraction of DNA undergoing
denaturation. Since neither intact protein nor *II is capable of
melting natural dsDNA, the kinetic block is at least partially
overcome. In the case of *III, there is no kinetic block, but under the
conditions employed (0.05 M NaCl, temperatures below
45 °C), the relatively low affinity of the protein for ssDNA
generates no Tm depression. So in addition to
overcoming the kinetic block, there is an apparent increase in the
noncooperative binding affinity of *III (and *II) for single-stranded
DNA. Along with the effect on hyperchromicity, the observed
Tm was seen to increase with increasing levels of
full-length, *II, or *III protein.
We can think of two explanations for these effects. One possibility is
that as *I brings about the melting of the double helix, the newly
formed single strands are now free to bind the other forms of the
protein. The *I monomers can rearrange themselves such that there is
available free single-stranded DNA to which the other protein can bind.
In a sense, *I serves as the "engine" for strand separation. This
explanation, however, does not account for the potentiation of melting
activity by *II and *III, which bind noncooperatively and more weakly
(by three or more orders of magnitude) than intact protein.
Alternatively, *I may directly interact with any of the other three
forms of the protein, in effect achieving a mingling of (at least two)
different forms of the protein while bound to ssDNA. This would be
energetically more favorable than *I alone binding to ssDNA, since
there will be additional protein-DNA and protein-protein interactions
resulting from the mingling. The inhibitory effect of double-stranded
DNA on the proteolysis of *III in the presence of *I can be explained by the additional binding of core domain to ssDNA formed upon melting
of the double helix. The overall affinity of the mixed protein-ssDNA
complex might be lower than for a pure *I-DNA complex, thus reducing
the Tm depression (as was observed).
Although this is the first report of cooccupancy on ssDNA of different
forms of gene 32 protein, it is known that intact 32 protein and *I can
each bind ssDNA simultaneously with T4 gene 59 protein (24). In the
case of intact protein, the C-terminal domain affects both the binding
of gene 59 protein to ssDNA and the morphology of 32 protein-59 protein
complexes (24). In the present work, although the C-terminal domain
might modulate the affinity for DNA, the protein-protein interactions
that occur are clearly dependent on the N-terminal domain.
Given the involvement of the C domain in both heterotypic
protein-protein association and modulation of DNA melting, it is interesting to speculate about a relationship between these two activities. Conceivably, binding of the C domain to another protein, e.g. T4 DNA polymerase, might prevent interaction of
residues in the C-terminal third of the protein with the DNA binding
site, and induce a conformational change that mimics the removal of this portion of the protein, thus promoting melting activity. With the
mingling effect that we have demonstrated, this activity would be
applied not only to the gene 32 protein directly in contact with the
polymerase but also to adjacent 32 proteins. A large number of
heterotypic protein-protein contacts have been demonstrated for 32 protein (11, 12, 25), so the consequences of such an effect would be profound.