©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Stoichiometry of 2`,5`-Oligoadenylate-induced Dimerization of Ribonuclease L
A SEDIMENTATION EQUILIBRIUM STUDY (*)

(Received for publication, September 26, 1995; and in revised form, December 6, 1995)

James L. Cole Steven S. Carroll Lawrence C. Kuo

From the Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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)(3) 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)(3). 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.


INTRODUCTION

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.


MATERIALS AND METHODS

Oligoribonucleotides were obtained from The Midland Certified Reagent Company. Human ribonuclease L was expressed and purified as described previously (^1)and stored in 40% glycerol, 25 mM HEPES, pH 7.5, 100 mM KCl, 5.8 mM MgCl(2), and 5 mM DTT. (^2)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(2)) 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 times 10^4M cm (average of 4 determinations). The concentrations of 2`,5`-adenosine trimer (2,5A(3)) and p2,5A(3), its 5`-monophosphate derivative, were measured spectrophotometrically using an extinction coefficient of = 4.59 times 10^4M 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(0) is the protein concentration at the reference distance r(0), M(w) 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(3) (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]CUC(8) as the RNA substrate in the same buffer used in the incubation plus 1.2 mM ATP but without any additional p2,5A(3). 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.


RESULTS AND DISCUSSION

Fig. 1shows the concentration profiles of ribonuclease L (233 nM loading concentration) in the absence and presence of 400 nM p2,5A(3). 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(3). 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 Kfor 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 (circle) and in the presence (box) of 400 nM p2,5A(3). 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(3), K, has been estimated to be less than 10 nM under conditions similar to those employed for the sedimentation experiments. (^3)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(w) is given by,

where M(1) is the monomer molecular weight. Thus, F = (M(w)/M(1)) - 1. Values of M(w) were obtained by fitting sedimentation equilibrium profiles obtained at various activator to ribonuclease L ratios using . The value of M(1) was fixed at the ribonuclease L monomer molecular weight of 83,400. The stoichiometry of activator binding-induced dimerization was obtained by fitting the Fversus [activator] data to the binding equation for identical and independent binding sites,

where P(0) is the molar concentration of ribonuclease L subunits, R is the number of activator binding sites/subunit, and L(0) is concentration of activator added. In the limit where P(0)R + L(0) K, reduces to

Fig. 2shows the data for two titrations of the fraction dimeric ribonuclease L versus [p2,5A(3)] 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(3) molecule per ribonuclease L monomer.


Figure 2: Titration of p2,5A(3)-induced dimerization of ribonuclease L. 233 nM (circle) or 500 nM (box) ribonuclease L were incubated with the indicated concentrations of p2,5A(3) 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(3) was determined by incubating ribonuclease L with various concentrations of activator followed by dilution into a reaction mixture containing 2 µM of the substrate CUC(8). 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 CUC(8)versus [p2,5A(3)]. Fitting of these data to returns a best fit value of R = 1.15 ± 0.14. (^4)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(3). Ribonuclease L (200 nM) was incubated with p2,5A(3) (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]CUC(8) as substrate but did not include additional p2,5A(3) 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(3), 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(3) and 2,5A(4), also induce dimerization of ribonuclease L. Fig. 4shows a titration of fraction dimer versus [2,5A(3)] performed at two protein concentrations: 300 and 800 nM. As in the case of p2,5A(3) (Fig. 2) the data sets overlay closely and fit well to , indicating that P(0)R + L(0) 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(3)-induced dimerization of ribonuclease L. 300 nM (circle) or 800 nM (box) ribonuclease L were incubated with the indicated concentrations of 2,5A(3) 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(3). Carroll et al.^1 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`-CUUC(7)-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(2). 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(2)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(2)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 EA (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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
Carroll, S. S., Chen, E., Viscount, T., Geib, J., Sardana, M., Gehman, J., and Kuo, L. C.(1996) J. Biol. Chem.271, in press.

(^2)
The abbreviations used are: DTT, dithiothreitol; p2,5A(3), 5`-monophosphate 2`,5`-adenosine trimer; 2,5A(3), 2`,5`-adenosine trimer; 2,5A(4), 2`,5`-adenosine tetramer.

(^3)
S. S. Carroll, unpublished observations.

(^4)
In contrast to the sedimentation results, activity data sets obtained at several protein (100-200 nM) and substrate (1-2 µM) concentrations do not precisely overlay. The value of R may be weakly perturbed by the presence of substrate such that R tends to increase as the enzyme concentration decreases. Possibly, substrate perturbs the binding of activator. The data presented in Fig. 4were obtained at the highest enzyme concentration that is experimentally accessible.


ACKNOWLEDGEMENTS

We thank Tracy Viscount for growing baculovirus-infected insect cells and James Geib for purification of ribonuclease L. We also thank members of the Reversible Associations in Structural and Molecular Biology group for suggesting methods to maintain low UV buffer absorption in samples containing DTT.


REFERENCES

  1. Kerr, I. M., and Brown, R. E. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 256-260 [Abstract]
  2. Floyd-Smith, G., Slattery, E., and Lengyel, P. (1981) Science 212, 1030-1032 [Medline] [Order article via Infotrieve]
  3. Wreschner, D. H., McCauley, J. W., Skehel, J. J., and Kerr, I. M. (1981) Nature 289, 414-417 [Medline] [Order article via Infotrieve]
  4. Zhou, A., Hassel, B. A., and Silverman, R. H. (1993) Cell 72, 753-765 [Medline] [Order article via Infotrieve]
  5. Dong, B., and Silverman, R. H. (1995) J. Biol. Chem. 270, 4133-4137 [Abstract/Free Full Text]
  6. Wyman, J., and Gill, S. J. (1990) in Binding and Linkage: Functional Chemistry of Biological Macromolecules , pp. 203-236, University Science Books, Mill Valley, CA
  7. Cohn, E. J., and Edsall, J. T. (1943) in Proteins, Amino Acids and Peptides as Ions and Dipolar Ions (Cohn, E. J., and Edsall, J. T., eds), p. 157, Rheinhold, New York
  8. Durchschlag, H. (1986) in Thermodynamic Data for Biochemistry and Biotechnology (Hinz, H. J., ed) pp. 45-128, Springer-Verlag, New York
  9. Johnson, M. L., Correia, J. J., Yphantis, D. A., and Halvorson, H. R. (1981) Biophys. J. 36, 575-588 [Abstract]
  10. Dong, B., Xu, L., Zhou, A., Hassel, B. A., Lee, X., Torrence, P. F., and Silverman, R. H. (1994) J. Biol. Chem. 269, 14153-14158 [Abstract/Free Full Text]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.