Stoichiometry of 2′,5′-Oligoadenylate-induced Dimerization of Ribonuclease L

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 M, 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.Ribonuclease L is an enzyme involved in the interferon pathway (1). The enzyme is activated upon binding of adenosine oligomers linked 2Ј to 5Ј to cleave viral and cellular RNAs at the 3Ј-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 METHODSOligoribonucleotides 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 BioRad 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 Ϯ 0.87 ϫ 10 4 M Ϫ1 cm Ϫ1 (average of 4 determinations). The concentrations of 2Ј,5Ј-adenosine trimer (2,5A 3 ) and p2,5A 3 , its 5Ј-monophosphate derivative, were measured spectrophotometrically using an extinction coefficient of ⑀ 260 ϭ 4.59 ϫ 10 4 M Ϫ1 cm Ϫ1 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, , was calculated to be 0.725...

A Bipartite Model of 2-5A-dependent RNase L

“…Binding of 2-5A could cause a conformational change in the enzyme that releases the inhibitory effect of the ankyrin repeats leading to both an unmasking of the ribonuclease and interaction domains near the C terminus. Previous studies involving multiple biochemical and biophysical methods established that the binding of 2-5A to monomers of RNase L induces the formation of RNase L homodimers (7,8). However, additional studies will be required to determine if dimerization is actually required for ribonuclease activity.…”

“…The proposed biological functions of RNase L includes involvement in the antiviral and antiproliferative mechanisms of interferon action and in the more general control of RNA decay (5,6). The human RNase L is a 741-amino acid protein that forms homodimers upon binding 2-5A (7,8). Activation of human RNase L requires 2-5A molecules containing at least three 2Ј,5Ј-adenylyl residues, such as pA(2Јp5ЈA) 2 , and the binding of 2-5A to RNase L, K D ϭ 40 pM, is highly specific (9,10).…”

Diverse functions of RNase L and implications in pathology

“…The dimerization and activation of RNase L requires a molar ratio of 1:1 between RNase L and 2-5A [33,36,37]. The C-terminal half of RNase L is homologous with the kinase/endoribonuclease, IRE1p, which functions in the unfolded protein response (UPR) in Saccharomyces cerevisiae, Caenorhabditis elegans and vertebrates including Homo sapiens [34,38,39].…”

A scientific journey through the 2-5A/RNase L system

“…Studies by the groups of Robert Suhadolnik [57,58] and Bernard LeBleu [59,60] described up-regulation of the 2-5A system and accumulation of a 37 kDa polypeptide from RNase L in PBMC of patients with chronic fatigue syndrome. In addition, Lawrence Kuo's group at Merck Research Laboratories performed outstanding enzymology and biophysical studies of RNase L [61][62][63][64].…”