Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2′–5′-oligoadenylate (2–5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293–15299). Sequence comparison of this region among the different isozymes of 2–5(A) synthetases revealed that the residues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present at 331, 332, and 333 are important for activity but the proline at position 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants were slightly active, and the three single mutants were partially active. The triple mutant was further characterized for delineating the nature of its defect. The mutant protein was enzymatically inactive irrespective of whether it was synthesized in rabbit reticulocyte lysate, Escherichia coli or Trichoplusia niinsect cells. It could bind double-stranded RNA and ATP as efficiently as the wild type protein. It was, however, defective in oligomerization. Gel filtration and sedimentation velocity analyses of in vitro synthesized proteins revealed that the wild type protein, but not the triple mutant, formed tetramers. The tetrameric fraction, but not the monomeric fraction of the wild type protein was enzymatically active. The failure of the triple mutant to participate in homomeric protein-protein interaction was confirmed by in vivo assays in insect cells. These results indicate that tetramerization of the protein is required for the enzymatic activity of the small 2–5(A) synthetases. Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2′–5′-oligoadenylate (2–5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293–15299). Sequence comparison of this region among the different isozymes of 2–5(A) synthetases revealed that the residues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present at 331, 332, and 333 are important for activity but the proline at position 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants were slightly active, and the three single mutants were partially active. The triple mutant was further characterized for delineating the nature of its defect. The mutant protein was enzymatically inactive irrespective of whether it was synthesized in rabbit reticulocyte lysate, Escherichia coli or Trichoplusia niinsect cells. It could bind double-stranded RNA and ATP as efficiently as the wild type protein. It was, however, defective in oligomerization. Gel filtration and sedimentation velocity analyses of in vitro synthesized proteins revealed that the wild type protein, but not the triple mutant, formed tetramers. The tetrameric fraction, but not the monomeric fraction of the wild type protein was enzymatically active. The failure of the triple mutant to participate in homomeric protein-protein interaction was confirmed by in vivo assays in insect cells. These results indicate that tetramerization of the protein is required for the enzymatic activity of the small 2–5(A) synthetases. 2′–5′-Oligoadenylate (2–5(A)) 1The abbreviations used are: 2–5(A), 2′–5′ oligoadenylates; dsRNA, double-stranded RNA; PKR, protein kinase, RNA-activated; poly(I)·poly(C), polyinosinic acid·polycytidylic acid; TM, triple mutant; WT, wild type. synthetases are a family of enzymes that are induced by interferons (1Vilcek J. Sen G.C. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. Lippincott-Raven Publishers, Philadelphia1996: 375-399Google Scholar, 2Hovanessian A. J. Interferon Res. 1991; 11: 199-205Crossref PubMed Scopus (149) Google Scholar). These enzymes require double-stranded RNA as a co-factor and they polymerize ATP into 2–5(A), which activates the latent ribonuclease, RNase L. In an interferon-treated cell, virus infection often causes the production of dsRNA, which activates 2–5(A) synthetases leading to the production of 2–5(A), activation of RNase L, and degradation of cellular RNA. The 2–5(A) synthetase/RNase L pathway is responsible for mediating interferon's antiviral effect against picornavirus, and the same pathway has also been implicated in the antigrowth effects of interferon. Another interferon-induced enzyme that requires dsRNA as a co-factor is the protein kinase, PKR (1Vilcek J. Sen G.C. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. Lippincott-Raven Publishers, Philadelphia1996: 375-399Google Scholar, 3Maran A. Maitra R.K. Kumar A. Dong B. Xiao W. Li G. Williams B.R. Torrence P.F. Silverman R.H. Science. 1994; 265: 789-792Crossref PubMed Scopus (210) Google Scholar, 4Samuel C.E. J. Biol. Chem. 1993; 268: 7603-7606Abstract Full Text PDF PubMed Google Scholar). The dsRNA-binding domain of PKR overlaps with its dimerization domain (5Patel R.C. Stanton P. McMillan N.M.J. Williams B.R.G. Sen G.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8283-8287Crossref PubMed Scopus (144) Google Scholar). This domain contains two dsRNA-binding motifs that are also present in a number of other dsRNA-binding proteins (6McCormack S.J. Thomis D.C. Samuel C.E. Virology. 1992; 188: 47-56Crossref PubMed Scopus (154) Google Scholar, 7Feng G.S. Chong K. Kumar A. Williams B.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5447-5451Crossref PubMed Scopus (158) Google Scholar, 8Katze M.G. Wamback M. Wong M.L. Garfinkel M. Meurs E. Chong K. Williams B.R. Hovanessian A.G. Barber G.N. Mol. Cell. Biol. 1991; 11: 5497-5505Crossref PubMed Scopus (174) Google Scholar, 9Green S.R. Mathews M.B. Genes Dev. 1992; 6: 2478-2490Crossref PubMed Scopus (218) Google Scholar, 10Patel R.C. Sen G.C. J. Biol. Chem. 1992; 267: 7671-7676Abstract Full Text PDF PubMed Google Scholar, 11St. Johnston D. Brown N.H. Gall J.G. Jantsch M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10979-10983Crossref PubMed Scopus (486) Google Scholar). 2–5(A) synthetases, however, do not contain this motif and the critical residues responsible for dsRNA binding by these proteins have not yet been identified. There are three size classes of 2–5(A) synthetases: large, medium, and small (2Hovanessian A. J. Interferon Res. 1991; 11: 199-205Crossref PubMed Scopus (149) Google Scholar). The different isozymes are structurally related, and there is strong sequence conservation across the species (12Ghosh S.K. Kusari J. Bandyopadhyay S.K. Samanta H. Kumar R. Sen G.C. J. Biol. Chem. 1991; 266: 15293-15299Abstract Full Text PDF PubMed Google Scholar). The large human synthetase is 100 kDa, and the medium synthetase is 69 kDa. The small synthetases have molecular weights ranging from 40 to 46 kDa (13Marie I. Hovanessian A. J. Biol. Chem. 1992; 267: 9933-9939Abstract Full Text PDF PubMed Google Scholar). There are two human small synthetases, E16 and E18, that are the products of alternatively spliced mRNAs of the same gene (14Chebath J. Benech P. Revel M. Vigneron M. Nature. 1987; 330: 587-588Crossref PubMed Scopus (219) Google Scholar). There are three murine small synthetases: 9-2 and 3-9 isozymes are the products of two alternatively spliced mRNA (12Ghosh S.K. Kusari J. Bandyopadhyay S.K. Samanta H. Kumar R. Sen G.C. J. Biol. Chem. 1991; 266: 15293-15299Abstract Full Text PDF PubMed Google Scholar), whereas L3 is encoded by a separate gene (15Ichii Y. Fukunaga R. Shiojiri S. Sokawa Y. Nucleic Acids Res. 1986; 14: 10117Crossref PubMed Scopus (46) Google Scholar). The different isozymes reside in different subcellular compartments. Their specific subcellular locations are sometimes dictated by their specific post-translational modifications. The small synthetases can form tetramers, whereas the medium synthetase exists as a dimer (16Marie I. Svab J. Robert N. Galabru J. Hovanessian A.G. J. Biol. Chem. 1990; 265: 18601-18607Abstract Full Text PDF PubMed Google Scholar). We cloned the cDNA of the murine 9-2 isozyme and have been investigating its structure-function relationship (12Ghosh S.K. Kusari J. Bandyopadhyay S.K. Samanta H. Kumar R. Sen G.C. J. Biol. Chem. 1991; 266: 15293-15299Abstract Full Text PDF PubMed Google Scholar, 17Desai S.Y. Patel R.C. Sen G.C. Malhotra P. Ghadge G.D. Thimmapaya B. J. Biol. Chem. 1995; 270: 3454-3461Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We have reported that the loss of as little as 9 residues from the amino terminus of the protein inactivates it (18Ghosh A. Desai S.Y. Sarkar S.N. Ramaraj P. Ghosh S.K. Bandyopadhyay S. Sen G.C. J. Biol. Chem. 1997; 272: 15452-15458Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The crucial residue within this region is proline at the 7th position. A mutant, P7Q, is enzymatically inactive. However, it can bind dsRNA and ATP, and the mutant protein can tetramerize like the wild type protein. In this report, we have investigated the properties of another class of 9-2 mutants which carry mutations near the carboxyl terminus. We have previously shown that a C-terminally truncated protein containing residues 1–344 is enzymatically active, but another deletion mutant containing residues 1–320 is inactive (12Ghosh S.K. Kusari J. Bandyopadhyay S.K. Samanta H. Kumar R. Sen G.C. J. Biol. Chem. 1991; 266: 15293-15299Abstract Full Text PDF PubMed Google Scholar). In this study, we have identified specific residues present between residues 320 and 344 that are required for enzymatic activity. A triple mutant containing alanine substitutions at position 331, 332, and 333 was enzymatically inactive. The mutant protein could bind dsRNA and ATP, but it failed to oligomerize. These results indicate that tetramerization of the protein is needed for its enzyme activity. Poly(I)·poly(C)-agarose was from Pharmacia Biotech Inc., polyethyleneimine cellulose plates were from EM Scientific and [α-32P]ATP and [35S]cysteine were from NEN Life Science Products. ATP-agarose containing adenosine 5′-γ-4-aminophenyl triphosphate was from Sigma and RNase inhibitor, restriction enzymes, and other fine chemicals were from Boehringer Mannheim. TNT, a DNA-driven in vitro translation system was purchased from Promega, and anti-FLAG M2 monoclonal antibody Sepharose was purchased from Eastman Kodak Co. Sephacryl S300 was a product of Pharmacia. Reagents for preparing recombinant baculoviruses and for expression of proteins in insect cells were purchased from Life Technologies, Inc. Immobilon polyvinylidene difluoride membrane was from Millipore, peroxidase-conjugated antibody was from Boehringer Mannheim. Enhanced chemiluminescence kit was purchased from Amersham. The 9-2 cDNA clone of 2–5(A) synthetase was mutagenized to replace residues 330, 331, 332, and 334 with alanines singly or in combination. The mutations were introduced in antisense primers used for polymerase chain reactions. The antisense primer also contained a DraII site present 3′ to the mutation sites in the 9-2 cDNA. The sense primer was located 5′ to an upstream HindIII site. Products from polymerase chain reactions were filled in using Klenow DNA polymerase and digested with HindIII and DraII. The fragments containing the mutations replaced the corresponding HindIII-DraII fragment of wild type 9-2 cDNA clone in pGEM7Zf(−). The newly synthesized portions of the mutant cDNAs were completely sequenced to confirm the presence of the desired mutations and the absence of any other mutation. Wild type and mutant hexahistidine-tagged synthetases were expressed in vitrousing the TNT rabbit reticulocyte lysate and [35S]cysteine. The products were analyzed as described before (18Ghosh A. Desai S.Y. Sarkar S.N. Ramaraj P. Ghosh S.K. Bandyopadhyay S. Sen G.C. J. Biol. Chem. 1997; 272: 15452-15458Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Bacterial expression of the hexahistidine-tagged triple mutant and its partial purification were done as described for the wild type protein (17Desai S.Y. Patel R.C. Sen G.C. Malhotra P. Ghadge G.D. Thimmapaya B. J. Biol. Chem. 1995; 270: 3454-3461Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). 2–5(A) synthetase assay and analysis of the products were done as described before (18Ghosh A. Desai S.Y. Sarkar S.N. Ramaraj P. Ghosh S.K. Bandyopadhyay S. Sen G.C. J. Biol. Chem. 1997; 272: 15452-15458Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 19Mory Y. Vaks B. Chebath J. J. Interferon Res. 1989; 9: 295-304Crossref PubMed Scopus (18) Google Scholar) except that the incubations were for 45 min. Poly(I)·poly(C)-agarose chromatography and ATP-agarose chromatography were done as before (18Ghosh A. Desai S.Y. Sarkar S.N. Ramaraj P. Ghosh S.K. Bandyopadhyay S. Sen G.C. J. Biol. Chem. 1997; 272: 15452-15458Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The conditions for this analysis were the same as described before (18Ghosh A. Desai S.Y. Sarkar S.N. Ramaraj P. Ghosh S.K. Bandyopadhyay S. Sen G.C. J. Biol. Chem. 1997; 272: 15452-15458Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In vitro translated proteins were analyzed at room temperature on a 1 × 50-cm Sephacryl S300 column. The column was equilibrated with a buffer containing 20 mm Tris-Cl, pH 7.5, 450 mm mm mm and of in vitro translation product was with of the buffer and to the column. were in of the were by of in the presence of of The were in buffer and analyzed by The 9-2 protein were by The proteins were by 9-2 protein was synthesized in It was size by filtration as described and the tetrameric and the monomeric were The were with anti-FLAG antibody to Sepharose in the filtration buffer for 1 at The Sepharose were with 20 mm Tris-Cl, pH 7.5, and and at portions of the were analyzed by for the of of tetrameric and monomeric to the were for with for activity The products were analyzed and using described were analyzed by and into a polyvinylidene difluoride The membrane was in at for by at in containing of a monoclonal antibody against 2–5(A) synthetase M. M. K. Sokawa Y. J. Interferon Res. 1995; PubMed Scopus (18) Google Scholar). The membrane was for with three of containing It was with with at a for 1 at with The membrane was for with of containing 20 and with chemiluminescence for 1 before to a Wild type was purchased from Life Technologies, Inc. and recombinant were in of S. and of proteins were expressed in cells The cells were at in insect containing that has been for insect and and Technologies, The desired cDNA of hexahistidine-tagged 9-2 proteins were cloned in at the were and the DNA was used to cells. containing recombinant were identified by the loss of activity. molecular DNA was and used to S. cells using The were and as virus at were by assays using the same cells. protein cells were with the recombinant virus at a of infection of were 40 three in and at by for using in a buffer containing 20 mm Tris-Cl, pH 7.5, mm 1 mm 1 mm 1 mm 1 1 and The was at × for and the was at for further cells were with recombinant singly or in two were of were were as described of proteins present in these was done as described before (18Ghosh A. Desai S.Y. Sarkar S.N. Ramaraj P. Ghosh S.K. Bandyopadhyay S. Sen G.C. J. Biol. Chem. 1997; 272: 15452-15458Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) with the modifications. The anti-FLAG Sepharose were in for at with This was by with the at of protein, for 1 at with The were before analysis of the proteins by and The murine 9-2 isozyme of 2–5(A) synthetase has deletion we have previously shown (12Ghosh S.K. Kusari J. Bandyopadhyay S.K. Samanta H. Kumar R. Sen G.C. J. Biol. Chem. 1991; 266: 15293-15299Abstract Full Text PDF PubMed Google Scholar) that the protein can truncated from the carboxyl terminus residues a loss of enzyme activity. deletion of residues however, the protein of specific residues we the amino sequence of this region with of the corresponding of other isozymes of 2–5(A) synthetase the small synthetases, the residues are completely among 9-2 and 3-9 and human E16 and The isozyme on the other has partial The same is for the medium synthetase L3 and were the isozymes and residues 330, 331, and were in these we to on their in 9-2 activity. We residue 333 to this it was not in L3 and we have previously shown that in 9-2 it can to a loss of activity. Alanine-scanning mutagenesis was done to residues singly or in for their in 9-2 enzyme activity. The single mutants were partially The 330 mutant was as as the protein that the proline residue at that position is not The other three single mutants were as as the protein. mutations of residues 331, 332, or 333 of we to on these double the three mutations in were the single three mutations were the triple mutant was completely inactive. We to that the loss of activity of the is not a of its in vitro in the reticulocyte We have previously shown that the 9-2 protein can expressed in E. and the protein has enzyme activity (17Desai S.Y. Patel R.C. Sen G.C. Malhotra P. Ghadge G.D. Thimmapaya B. J. Biol. Chem. 1995; 270: 3454-3461Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The system was used to the protein as and of partially and proteins were for enzyme activity. The was also inactive A and We have a expression system for the 9-2 protein in insect cells. Bandyopadhyay, A. S. N. and G. C. Sen, large of proteins can synthesized in insect cells and their post-translational are to in this system is sometimes to the expression shown in the and the 9-2 proteins were highly expressed in insect cells with the corresponding the 9-2 with the not the production of any protein. the protein was highly but the protein enzymatic activity. These results demonstrated that the was inactive irrespective of its which rabbit reticulocyte lysate, E. and insect cells. we that the was we to its other We used and ATP-agarose as to the of the to bind to its and shown in the could bind to as efficiently as the protein. our of the in protein to the and proteins to ATP-agarose we could not any of the with to its interaction with the and the we investigated the of the We have previously used sedimentation velocity analysis to that in vitro synthesized 9-2 protein can form (18Ghosh A. Desai S.Y. Sarkar S.N. Ramaraj P. Ghosh S.K. Bandyopadhyay S. Sen G.C. J. Biol. Chem. 1997; 272: 15452-15458Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). our which a for of of the 9-2 protein as a However, for the TM, the tetrameric was not This that the induced mutations could have the of the protein. confirm this we a filtration to the used by Hovanessian and (16Marie I. Svab J. Robert N. Galabru J. Hovanessian A.G. J. Biol. Chem. 1990; 265: 18601-18607Abstract Full Text PDF PubMed Google Scholar) to that small synthetases as tetramers. In vitro translated proteins were on a Sephacryl S300 proteins in different were by and the 9-2 were by shown in the wild type protein was into size was at the monomeric another was at the position of a and there was a at the tetrameric position of kDa. In the tetrameric was for the triple mutant; this protein was filtration and proteins were analyzed by chromatography on Sephacryl The positions of the different molecular are at the protein present in fraction is as of analysis of the in vitro synthesized proteins that the is defective in we to this further by using the mutant protein expressed in insect cells. In we used a assay that in vivo homomeric interaction of the 9-2 protein. were with two a 9-2 gene and another a or 9-2 were with antibody to the Sepharose were and the proteins were by and by using a synthetase The and the proteins could by their in of the and proteins were synthesized in cells for and from cells with single the protein to the it contained the and with the protein with the protein of protein-protein the other the triple mutant failed to with the protein they were present in the same cells These results that the the to with and form a The results that the of activity of the is to its failure to form tetramers. that monomeric 9-2 protein is also whereas the tetrameric form is active. This was in the shown in A large of 9-2 protein was synthesized in and the tetrameric and monomeric were by containing the two were and the proteins were by binding to anti-FLAG antibody to Sepharose of the tetrameric and monomeric of the protein were for enzyme activity the a enzyme activity with of the protein The was, however, inactive at the These results for the that the 9-2 protein requires tetramerization for its enzymatic activity. analysis of the 9-2 protein in our studies the of the residues A truncated protein containing the residues 1–320 could bind dsRNA, but it not have enzyme activity (12Ghosh S.K. Kusari J. Bandyopadhyay S.K. Samanta H. Kumar R. Sen G.C. J. Biol. Chem. 1991; 266: 15293-15299Abstract Full Text PDF PubMed Google Scholar). The of residues to the carboxyl terminus the activity In the study, we have on this region of the protein with the of the crucial We of the among the different synthetases for of the 9-2 sequence with of the L3 and the proteins was The of residues within this region residues and we an mutagenesis to this region of the protein. we the proline at 330 to the crucial its mutation to alanine not the activity of the 9-2 protein. In mutations of at 331, at 332, and at 333 the enzyme activity The double mutants were active, and the triple mutant was completely inactive. identified a of mutation to completely the enzymatic activity. We to the mutant protein in three different rabbit reticulocyte lysate, E. and insect cells. The wild type protein expressed in three was active, but the was inactive irrespective of its These results that the introduced mutations a of the protein and not its interaction with post-translational enzymes, which are different in the three different of expression used by from the of the of the dsRNA (12Ghosh S.K. Kusari J. Bandyopadhyay S.K. Samanta H. Kumar R. Sen G.C. J. Biol. Chem. 1991; 266: 15293-15299Abstract Full Text PDF PubMed Google Scholar) and sites on the protein N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (31) Google Scholar), the dsRNA and ATP as efficiently as the wild type protein. The mutant protein, however, was defective in tetramers. This that the tetrameric is the enzymatically The was by our of tetrameric and monomeric proteins that the tetrameric fraction was enzymatically active. Hovanessian and (16Marie I. Svab J. Robert N. Galabru J. Hovanessian A.G. J. Biol. Chem. 1990; 265: 18601-18607Abstract Full Text PDF PubMed Google Scholar) that the small synthetases as tetramers, and the medium synthetase exists as a the medium synthetase has two of which has strong with the small synthetases (13Marie I. Hovanessian A. J. Biol. Chem. 1992; 267: 9933-9939Abstract Full Text PDF PubMed Google Scholar), they that for isozymes the is formed by the of The are by in the of the small synthetases, whereas two of the medium containing two are results this we that the same introduced to the protein, also that protein by its This to We do not have yet to the residues oligomerization. a residue is in the is not in the The is of the that of the wild type protein, as by size and protein-protein interaction is not by the presence of as or the mutant was partially active, that the residue is not the failed to in the protein-protein interaction it to that the residues reside at the site. The introduced mutations could have the of the protein in a that the at a different of the protein These the pathway to is can the dimerization of the protein by further dimerization of the The of as is in the results shown in the wild type protein, there was a at the position of a the mutant, there was at the position of the the dimer was it that the triple mutant can form but not tetramers. In that it in the protein-protein interaction The could to the different conditions used for the two The protein interaction assay used a to and that have been a for interaction of the mutant protein. assays to in the to further The presence of in the filtration analysis of the triple mutant that it to form that at a as with the The enzymatically inactive triple mutant for the cellular of 2–5(A) synthetases. it not with the wild type protein, it not its activity in S. N. W. S. Bandyopadhyay, and G. C. Sen, the mutant not as a in It however, as a for dsRNA in cells. It to can in a the triple the triple mutant, enzymatically a for cellular proteins that with 2–5(A) synthetases in The wild type protein is for that its expression causes cellular We Sokawa for the synthetase monoclonal antibody and for
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