(Received for publication, September 26, 1995; and in revised form, December 6, 1995)
From the
Ribonuclease L is an endoribonuclease that is activated by
binding of 2`,5`-linked oligoadenylates. Activation of ribonuclease L
also induces dimerization. Here, we demonstrate using equilibrium
sedimentation that dimerization requires the binding of one
5`-monophosphate 2`,5`-(adenosine) molecule per
ribonuclease L monomer. No dimerization was observed in the absence of
activator up to a protein concentration of 18 µM,
indicating that unliganded enzyme is unable to dimerize or the
association is very weak. In parallel with dimerization, enzymatic
activity is also maximized at a 1:1 activator: ribonuclease L
stoichiometry. The same stoichiometry for dimerization is observed
using a nonphosphorylated activator 2`-5`-(adenosine)
.
Adenosine triphosphate or RNA oligonucleotide substrates do not induce
dimerization. The observed stoichiometry supports a model for
ribonuclease L dimerization in which activator binds to monomer, which
subsequently dimerizes.
Ribonuclease L is an enzyme involved in the interferon pathway (1) . The enzyme is activated upon binding of adenosine oligomers linked 2` to 5` to cleave viral and cellular RNAs at the 3`-side of UpNp sequences(2, 3) . Human ribonuclease L has been cloned and overexpressed in a baculovirus system(4) . Recently, it was demonstrated by biochemical methods that ribonuclease L exists as a monomer in solution but is dimerized in the presence of activator, suggesting that the catalytically active form of ribonuclease L is a homodimer(5) . However, the stoichiometry and affinity for the activator-induced dimerization are not known. As an initial step in the development of a thermodynamic model for the activation of ribonuclease L we have employed equilibrium sedimentation to define the stoichiometry of activator-induced dimerization of the enzyme.
Oligoribonucleotides were obtained from The Midland Certified
Reagent Company. Human ribonuclease L was expressed and purified as
described previously ()and stored in 40% glycerol, 25 mM HEPES, pH 7.5, 100 mM KCl, 5.8 mM MgCl
, and 5 mM DTT. (
)In order to
reduce the UV absorbance due to oxidized DTT, the sample buffer (11
mM HEPES, pH 7.5, 104 mM KCl, 5.8 mM MgCl
) was purged of oxygen by bubbling with argon
prior to adding 1 mM DTT.
Ribonuclease L was equilibrated
into the sample buffer using Bio-Rad Biospin 6 spin columns. Protein
concentration was measured spectrophotometrically. The molar extinction
coefficient at 280 nm was determined by amino acid analysis to be 8.41
± 0.87 10
M
cm
(average of 4 determinations). The
concentrations of 2`,5`-adenosine trimer (2,5A
) and
p2,5A
, its 5`-monophosphate derivative, were measured
spectrophotometrically using an extinction coefficient of
= 4.59
10
M
cm
with an estimated standard deviation of 5%.
The uncertainties in the concentrations of enzyme and activator were
propagated in the final calculation of the uncertainty of the
stoichiometry of activator binding. The partial specific volume of
ribonuclease L, t;ex2html_html_special_mark_amp;ngr;, was calculated to be
0.725 at 25 °C using the method of Cohn and Edsall (7) and
adjusted for temperature(8) . The solvent density,
, was
measured to be 1.0066 at 4 °C using an Anton Paar DMA 48 density
meter. Samples were loaded into 6-channel (1.2-cm path) or 2-channel
(0.3-cm path) centrifuge cells under argon, and equilibrium analytical
centrifugation was performed at 4 °C using a Beckman XL-A
centrifuge. Scans were taken at 230 or 276 nm. At 230 nm there is
negligible contribution to the absorbance from RNA and
2`,5`-oligoadenylate derivatives. Equilibrium was judged to be achieved
by the absence of systematic deviations in a plot of the difference
between successive scans. Molecular weights were obtained by fitting
the data to the expression,
where C is the total protein
concentration, C
is the protein concentration at
the reference distance r
, M
is the weight average molecular weight, and
is the angular
velocity. Data analysis was performed using the nonlinear least-squares
programs NONLIN (9) and KaleidaGraph (Abelbeck Software).
For activity measurements ribonuclease L (50-200 nM)
was incubated on ice with p2,5A (50-600 nM)
in buffer containing 11.5 mM HEPES, pH 7.6, 104 mM KCl, 5.8 mM magnesium acetate, 5 mM DTT, and
0.2% polyethylene glycol 8000 for 30 min. Reactions (100 µl) were
initiated by addition of an aliquot of the incubation solution to a
reaction mixture containing 2 µM 5`-[
P]C
UC
as the
RNA substrate in the same buffer used in the incubation plus 1.2 mM ATP but without any additional p2,5A
. Aliquots (8
µl) were quenched after a 2-min reaction time with an equal volume
of gel load buffer. Products were separated by denaturing gel
electrophoresis and were quantified using a Molecular Dynamics
PhosphorImager.
Fig. 1shows the concentration profiles of
ribonuclease L (233 nM loading concentration) in the absence
and presence of 400 nM p2,5A. The data for the
sample in the absence of activator fit well to a single ideal species
model with a molecular weight of 83,800 ± 4,500, in excellent
agreement with the molecular weight of 83,400 deduced from the amino
acid sequence. Addition of excess activator results in an increase of
the molecular weight to 162,000 ± 8,000, which is close to the
value expected for a dimer of ribonuclease L (166,800). Thus, the
enzyme is monomeric in the absence and dimeric in the presence of
excess p2,5A
. These results confirm an earlier report (5) that activators induce dimerization of the ribonuclease L.
The present results also demonstrate that close to 100% of the
ribonuclease L in our preparation is competent for dimerization. In
separate experiments we observe that in the absence of activator
ribonuclease L remains completely monomeric up to protein
concentrations of 18 µM (data not shown), indicating that
ribonuclease L cannot dimerize in the absence of activator, or K
for dimerization is significantly
greater than 20 µM. Conversely, in the presence of
saturating activator, ribonuclease L is fully dimerized at a protein
concentration as low as 100 nM (data not shown), indicating
that K
for fully liganded ribonuclease L
is much less than 100 nM. Analytical ultracentrifugation
experiments cannot readily be performed at lower ribonuclease L
concentrations because of limited UV absorption of the enzyme.
Figure 1:
Sedimentation equilibrium of
ribonuclease L. A, absorption profiles (230 nm) of 233 nM ribonuclease L sedimented at 14,000 rpm, 4 °C in the absence
() and in the presence (
) of 400 nM p2,5A
. Solid lines are nonlinear
least-squares fit to the data to . The molecular weights
are 83,800 ± 4,500 kDa in the absence and 162,000 ± 8,000
in the presence of activator. B, residuals for the fits in A.
It is
important to define the stoichiometry of activator binding to
ribonuclease L in order to develop a thermodynamic model for the
activation/dimerization process and to interpret results from enzyme
kinetics studies. In kinetic experiments, the dissociation constant for
p2,5A, K
, has been estimated
to be less than 10 nM under conditions similar to those
employed for the sedimentation experiments. (
)Thus, the
stoichiometry for activator binding-induced dimerization can be
obtained by characterizing the dependence of the dimerization on the
molar ratio of activator to ribonuclease L monomers under conditions
where the concentration of enzyme is held much higher than K
and K
. In
the case of a monomer-dimer equilibrium, the relevant parameter to
characterize the stoichiometry of ligand-induced dimerization is the
weight fraction of dimer, F
, which is
given by,
where W is the weight
concentration of dimer and W
is the
weight concentration of monomer. For a monomer-dimer system, M
is given by,
where M is the monomer molecular weight.
Thus, F
= (M
/M
) - 1. Values of M
were obtained by fitting sedimentation
equilibrium profiles obtained at various activator to ribonuclease L
ratios using . The value of M
was
fixed at the ribonuclease L monomer molecular weight of 83,400. The
stoichiometry of activator binding-induced dimerization was obtained by
fitting the F
versus [activator] data to the binding equation for identical
and independent binding sites,
where P is the molar concentration of
ribonuclease L subunits, R is the number of activator binding
sites/subunit, and L
is concentration of activator
added. In the limit where P
R + L
K
, reduces to
Fig. 2shows the data for two titrations of the fraction
dimeric ribonuclease L versus [p2,5A]
performed at protein concentrations of 233 and 500 nM. The two
data sets overlay nicely and were simultaneously fit to to
obtain a best fit parameter of R = 1.07 ± 0.14.
Thus dimerization of ribonuclease L requires binding of one p2,5A
molecule per ribonuclease L monomer.
Figure 2:
Titration of p2,5A-induced
dimerization of ribonuclease L. 233 nM (
) or 500 nM (
) ribonuclease L were incubated with the indicated
concentrations of p2,5A
and analyzed by sedimentation
equilibrium under the same conditions as in Fig. 1. The solid line is a nonlinear least fit of the two data sets to with a best fit value of R = 1.07 ±
0.14.
The stoichiometry for the
activation of ribonuclease L by p2,5A was determined by
incubating ribonuclease L with various concentrations of activator
followed by dilution into a reaction mixture containing 2 µM of the substrate C
UC
. The activity assays
were performed immediately following dilution. Fig. 3shows a
plot of the dependence of the rate of ribonuclease L cleavage of the
substrate C
UC
versus [p2,5A
]. Fitting of these data to returns a best fit value of R = 1.15
± 0.14. (
)These data suggest that the predominant
active species for RNA hydrolysis is the dimer containing two bound
activator molecules.
Figure 3:
Stoichiometry of activation of
ribonuclease L by p2,5A. Ribonuclease L (200 nM)
was incubated with p2,5A
(50-600 nM) on ice
in reaction buffer as described under ``Materials and
Methods.'' Cleavage reactions were initiated by addition of 2
µM 5`-[
P]C
UC
as substrate but did not include additional p2,5A
so
that the same ratio of activator to enzyme was maintained in the
cleavage reaction as in the incubation. The rates are shown relative to
the average of the rates obtained at stoichiometries of
activator:ribonuclease L subunits greater than 1.0. The data were fit
to , which indicated that the maximal activity was reached
at a ratio of 1.15 ± 0.14 activator to ribonuclease L
monomer.
In addition to p2,5A, various
2`,5`-linked oligoadenylate derivatives are known to also serve as
activators of ribonuclease L and to induce dimerization. In agreement
with an earlier report(5) , we find that activators lacking a
5`-phosphate, 2,5A
and 2,5A
, also induce
dimerization of ribonuclease L. Fig. 4shows a titration of
fraction dimer versus [2,5A
] performed
at two protein concentrations: 300 and 800 nM. As in the case
of p2,5A
(Fig. 2) the data sets overlay closely and
fit well to , indicating that P
R + L
K
. The best fit value of R is
1.10 ± 0.14, which is most consistent with a 1:1
activator:ribonuclease L binding ratio. In contrast, Dong and Silverman (5) have suggested that the stoichiometry depends on the
identity of the activator. The origin of this discrepancy is not clear.
Figure 4:
Titration of 2,5A-induced
dimerization of ribonuclease L. 300 nM (
) or 800 nM (
) ribonuclease L were incubated with the indicated
concentrations of 2,5A
and analyzed by sedimentation
equilibrium under the same conditions as in Fig. 1. The solid line is a nonlinear least fit of the two data sets to giving a value of R = 1.10 ±
0.14.
Adenosine triphosphate is found to enhance the activity of
ribonuclease L in the presence of activators(10) . However, we
have found that ATP up to a concentration of 50 µM does
not induce dimerization and does not influence the dimerization induced
by binding of 2,5A. Carroll et al.
have recently kinetically defined several synthetic
oligoribonucleotide substrates. The effect of ribonuclease L substrates
on the dimerization was tested. Addition of 1 µM of the
substrate 5`-C
UUC
-3` (K
= 205 nM) to ribonuclease L does not induce
dimerization. Thus, either substrates do not bind in the absence of
activators or binding of substrate does not induce dimerization.
The
data presented here provide constraints on the mechanism of activation
of ribonuclease L. Dimerization of ribonuclease L may proceed via three
possible routes as shown in , where E is
ribonuclease L monomer and A is activator. In (a) dimerization occurs
prior to activator binding, whereas in (b) and (c) dimerization
requires prior binding of either one or two activators, respectively.
Note that these mechanisms are not mutually exclusive. We have not
found any evidence for the unliganded dimer, E.
However, we cannot completely exclude mechanism (a), since the
dimerization constant could be extremely weak. The observed
stoichiometry of 1:1 for ligand-induced dimerization indicates that E
A does not accumulate to a significant extent,
which would tend to reduce R toward 0.5. Thus, if mechanism
(a) were operative, then the binding of the two activators would have
to be a highly cooperative. Similarly, mechanism (b) would require that
binding the second activator molecular to E
A
occurs much more readily than to the monomer. Taken together, these
data suggest that dimerization of ribonuclease L likely proceeds via
mechanism (c).
E+EA E
A (b)
Ligand-linked oligomerization of proteins is a commonly observed biological regulatory mechanism which is analogous to allostery(6) . However, an additional feature in oligomerizing systems is the dependence on protein concentration. Thus, ligand binding measurements as a function of enzyme concentration will be useful to define the coupled equilibria depicted in . In concert with enzymatic activity measurements, these studies will allow a detailed description of the relationship between the ligation/association states of ribonuclease L and catalysis.