1 Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, Department of Biochemistry, University College of Science, University of Calcutta, 35, Ballygunge Circular Road, Calcutta 700019, West Bengal, India
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Abstract |
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Keywords: Chandipura virus/dimerization/phosphoprotein/phosphorylation/transcription
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Introduction |
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Analysis and characterization of 50 different temperature-sensitive (ts) mutants of CHP virus resulted in the identification of six complementation groups. The intragroup complementation observed among the CHP virus ts mutants suggested that the functional form of at least one of the virion proteins of CHP virus is a multimer (Gadkari and Pringle, 1980a,b
).
The 11 kb long genomic RNA, encapsidated by nucleocapsid protein (N-RNA), serves as the template for both replication and transcription (Masters and Banerjee, 1987). Transcription of this genome by viral encoded RNA polymerase produces one leader RNA of 49 bases along with five capped and polyadenylated messenger RNAs which code for five different structural proteins, the nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G) and the large protein (L) in sequential order and in decreasing amounts. The active transcriptional regime can be carried in a defined system consisting of three viral components: the template (N-RNA), RNA-dependent RNA polymerase (L) and the phosphoprotein (P).
The P protein (32.5 kDa) from both virion and infected cells is always in the phosphorylated form. The phosphoprotein P of CHP virus has been studied in great detail in recent years in our laboratory, mostly regarding its role in transcription. Comparison of the amino acid (aa) sequence of the CHP virus P protein and VSV (New Jersey and Indiana serotypes) revealed only 21% similarities with no consecutive stretches of more than four amino acids being identical between them. The P protein of CHP virus (293 aa) is longer than that of the VSV (NJ) strain (274 aa) and their sites of phosphorylation are also different (Masters and Banerjee, 1987; Chattopadhyay et al., 1997
). These apparent dissimilarities in CHP virus P protein and its ability to infect human prompted us to study the protein in greater detail. The simple composition and complex behaviour of P protein make it a very good candidate for structurefunction studies.
In different rhabdoviruses the P protein is found to be present in different phosphorylated forms in the matured virion particle (Barik and Banerjee, 1992b). Transcription activation of P has been claimed to be a two-step process. First the unphosphorylated P undergoes phosphorylation by a host kinase to form the protein P1 which then undergoes phosphorylation again by L-associated kinase (LAK) to form P2 (Barik and Banerjee, 1992b
); However, according to Gao and Lenard (1995a), this second phosphorylation may also be associated with a kinase of host origin. It has been found that the phosphorylation by the host kinase is sufficient for the transcription in vitro (Barik and Banerjee, 1992a
).
Previous studies in our laboratory demonstrated that unphosphorylated recombinant P protein expressed in bacteria was transcriptionally inactive. Activation required phosphorylation by a cellular protein kinase which was identified as casein kinase II (CKII) (Chattopadhyay and Chattopadhyay, 1994). Only CKII and no other protein kinases, viz. PKC and PKA tested in vitro, phosphorylates the bacterially expressed P protein (data not shown). The stoichiometry of phosphorylation in vitro reaches a maximum value of 1 mol of phosphate per mole of P protein. Loss of transcription-supporting ability of P protein on substitution of Ser62 by Ala showed that phosphorylation of Ser62 is necessary for transcription in vitro (Chattopadhyay et al., 1997
). The mutant P protein (Ser62Ala), when cloned in a eukaryotic expression vector under CMV promoter and expressed in VERO cells, was found to be unphosphorylated, whereas under similar condition the wild-type P protein undergoes phosphorylation (data not shown). All these observations conclusively prove that only CKII phosphorylates the P protein and this phosphorylation at Ser62 is essential and sufficient to confer full transcriptional activity.
The role of different cellular kinases is established in the transcription activation property of the phosphoprotein of the related VSV (Chattopadhyay and Banerjee, 1987). However, the mechanism of phosphorylation-dependent transcription activation is still an enigma. Using different biophysical approaches we have proved that there is a change in the protein conformation at the N-terminal half of the P protein of CHP virus after phosphorylation (Raha et al., 1999
). Gel filtration studies showed that the in vitro phosphoryated protein elutes at the position of dimer whereas the unphosphoryated protein elutes as monomer from the column with respect to its molecular weight (Chattopadhyay et al., 1997
). But the elution volume cannot be taken as a measure of accurate molecular weight as changes in shape also affect precise elution position.
In this work we explored the nature of the phosphorylation-dependent conformational alteration of the three-dimensional structure of the phosphoprotein P of CHP virus. We approached this question by measuring the change in the hydrophobic character of P protein after phosphorylation. We also monitored the conformational change due to phosphorylation by partial proteolytic digestion followed by SDSPAGE separation of the peptide fragments to generate a `fingerprint' that is the characteristic of a particular protein substrate having different tertiary structures. Recently, in the case of VSV (IND), the phosphorylated P protein was shown to be a trimer by His-tag dilution assay (Gao et al., 1996). Using the same approach, we have shown that P protein of CHP virus is a homodimer. Using different deletion mutants of CHP virus P protein, we have also proved the involvement of an N-terminal CKII-modified region in PP homodimerization. Finally, we conclude that P protein, expressed in a bacterial system, did not dimerize although it contains the N-terminal 146 amino acid residues. Hence, upon phosphorylation, the change in the N-terminal end conformation helps the P protein to undergo dimerization.
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Materials and methods |
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Escherichia coli BL21(DE3) was transformed with pET3aPC plasmid containing the P gene. Cells containing the appropriate plasmid were grown in a medium containing 100 µg/ml ampicillin and 0.2% glucose at 25°C. At O.D.590 = 0.5, isopropyl ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM. Cells were harvested and lysed with lysozyme in 50 mM TrisHCl (pH 8.0) containing 0.1% Triton X-100 in the presence of protease inhibitors and then treated with DNase. The lysate was centrifuged to remove the debris and supernatant was used for purification of P protein.
Purification of the untagged P protein from the lysate was carried out using a Q-Sepharose (Pharmacia Biotech) column pre-equilibrated with 50 mM TrisHCl (pH 8.0) containing 0.1% Triton X-100 and 1 mM EDTA. The protein was eluted at a 300 mM NaCl salt concentration using a 0500 mM gradient (Chattopadhyay et al., 1997).
Expression and purification of His-tagged P protein from bacteria
The P gene was subcloned in pET15b vector under T7 promoter (Studier et al., 1990). The recombinant protein was expressed as His-tagged at the N-terminal end. The protein was expressed in E.coli BL21(DE3) as described earlier and purified through Ni-NTA agarose beads (Novagen). The column was equilibrated with 50 mM TrisHCl (pH 8.0) containing 0.1% Triton X-100 and 500 mM NaCl. After charging the protein, the column was washed with the same buffer containing 60 mM imidazole and the protein was eluted with the same buffer containing 1 M imidazole. The eluted protein was dialyzed against 50 mM TrisHCl (pH 8.0) containing 0.1% Triton X-100 and 1 mM EDTA to remove imidazole. The tagged protein migrates more slowly than the untagged protein in 10% SDSPAGE, which is the basis of the His-tag dilution experiment.
Cloning, expression and purification of the different deletion mutants of P protein having N-terminal His-tag
The P protein of CHP virus is 293 amino acids long. From the pET3a-PC clone the P gene was released and different restriction enzyme based deletion mutants of the CHPP gene were constructed in bacterial expression vectors pRSET A, B, C (Stratagene) keeping the reading frame unchanged. All the mutant P proteins have the N-terminal His-tag. The expressed proteins were simultaneously purified using Ni-NTA agarose beads as mentioned earlier and checked by SDSPAGE followed by Western blotting with antibody raised against P protein (data not shown). A schematic diagram of the different truncated P proteins is shown in Figure 1. The deletion mutant P177 contains the N-terminal 77 amino acid residues, which includes Ser62, i.e. the CKII target site. The deletion mutant P47230 contains amino acid residues 47230 and this mutant also contains Ser62. The deletion mutants P137293 and P78293 contain the amino acid residues 137293 and 78293, respectively. Owing to the presence of highly acidic amino acid residues, the P177 mutant showed abnormal migration in SDSPAGE.
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The purified unphosphorylated P protein (both untagged and tagged and the truncated P proteins) was phosphorylated in vitro by human recombinant CKII (Boehringer-Mannheim) in a transcription buffer containing 50 µM ATP for 1h at 30°C. To check the phosphorylation status of the different deletion mutants of P protein of CHP virus, the purified deletion mutants were subjected to in vitro phosphorylation by CKII under the same conditions.
To check the phosphorylation by LAK, the bacterially expressed P protein was phosphorylated in vitro by CKII using cold ATP. The phosphorylation reaction was stopped using heparin. The phosphorylated P protein was then incubated with lysed virus in presence of heparin and [32P]ATP.
Sensitivity of the incorporated phosphate group to alkaline phosphatases
A 1 µg amount of recombinant P protein was phosphorylated in vitro by CKII using [32P]ATP. The reaction was stopped by adding 5 µg/ml (final concentration) of heparin. The reaction mixture was dialyzed against 50 mM TrisHCl (pH 8.0) for 3 h to remove excess ATP. One part of the reaction mixture was treated with 1 U of calf intestinal alkaline phosphatases (CIAP, GIBCO-BRL) and another part was incubated with 1 U of bacterial alkaline phosphatase (BAP, GIBCO-BRL), both at 37°C for 30 min. The reaction mixtures along with the untreated samples were analyzed by 10% SDSPAGE followed by autoradiography. To check the activity of the phosphatases, the P protein was phosphorylated with CKII in presence of cold ATP and dialyzed and was subjected to phosphorylation with the viral LAK in the presence of heparin (5 µg/ml final concentration). The purified viral particle was lysed in the lysis buffer containing 10 mM TrisHCl (pH 8.0), 5% (v/v) glycerol, 0.4 M NaCl, 1.85% Triton X-100, 0.6 mM DTT. After centrifugation the supernatant (5 µl) was used as a source of LAK to phosphorylate the CKII phosphorylated P protein in presence of [
32P]ATP. After 30 min of phosphorylation, the reaction mixture was divided into four fractions: one part of the sample was mixed with SDS sample buffer to stop the reaction, the second part was incubated at 37°C without any phosphatase and the other two parts were incubated with CIAP and BAP, respectively, at 37°C for 30 min. The samples were analyzed by 10% SDSPAGE followed by autoradiography.
Binding of P protein to Phenyl Sepharose column
Binding of proteins to a Phenyl Sepharose column matrix gives us an idea of the exposed hydrophobic surface area of the protein. The binding of both the unphosphorylated and phosphorylated P protein to a Phenyl Sepharose gel matrix was determined according to Yamamato (1991). A 25 µg amount of purified P protein (both unphosphorylated and in vitro phosphorylated) in 60 µl of binding buffer containing 50 mM TrisHCl (pH 8.0), 0.1 M NaCl and various concentrations of ethylene glycol were added to 30 µl of a suspension of Phenyl Sepharose equilibrated with the same buffer. Samples were incubated at 4°C for 30 min. The suspensions were centrifuged at 6000 g for 1 min and the supernatants were analyzed by 10% SDSPAGE. The amount of proteins was quantitated by densitometric scanning of Coomassie Brilliant Blue-stained gel in a Pharmacia LKB laser scanner using a calibration curve constructed with known amounts of P protein.
Protein fingerprinting by limited protease digestion
Amounts of 20 µg each of purified phosphorylated and unphosphorylated P protein were incubated at 37°C for 10 min with different concentrations of TPCK-trypsin and chymotrypsin and the reactions were stopped with 50 µg/ml TLCK and TPCK, respectively. The samples were analyzed by 16% SDSPAGE and stained with Coomassie Brilliant Blue.
His-tag dilution assay
Unphosphorylated P protein and N-terminal His-tagged unphosphorylated P protein were obtained from overexpression of pET3aPC and pET15PC clones, respectively. Untagged protein moves faster than the tagged protein in 10% SDSPAGE. To a constant amount of tagged protein (1 µg), increasing amount of untagged proteins were added and the mixtures were subjected to in vitro phosphorylation by CKII. Multimers possessing at least one tag subunit were recovered through Ni-binding resin. The different sets were analyzed by 10% SDSPAGE. The proteins in the gel were quantitated by laser densitometric scanning after Coomassie Brilliant Blue staining. Band intensities were related to standard concentrations of tagged and untagged P protein separately. The molar ratios of untagged to tagged protein in the purified complex were plotted against the weight ratios of the untagged to tagged protein in the original mixture.
To identify the region involved in PP oligomerization, the same experiment was performed in which the His-tagged full-length P protein was replaced with different deletion mutants of P protein having His-tag at the N-terminal end. The phosphorylation status of different truncated P proteins was checked by in vitro phosphorylation with CKII. A 1 µg amount of the purified untagged P protein was mixed with the different purified deletion mutants having the same concentration (1 µg). The different mixtures were phosphorylated in vitro by CKII and then passed through a column containing Ni-NTA agarose in order to purify the dimer containing at least one tagged subunit as well as the free tagged protein. The samples were then analyzed by 10% SDSPAGE.
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Results |
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To identify whether the CKII-incorporated phosphate group is accessible to alkaline phosphatases, the bacterially expressed P protein was subjected to in vitro kinasing by CKII using [32P]ATP. The reactions were stopped by heparin followed by incubation with CIAP and BAP. An autoradiogram of the CIAP- and BAP-treated phosphorylated P protein shows the same intensities as the untreated one (Figure 2A
, lanes 14), which indicates that CKII irreversibily incorporated phosphate group into protein in such a way that it became inaccessible to the phosphatases used in the experiment. As a control, we phosphorylated the P protein by LAK in presence of heparin (to nullify the possibility of phosphorylation by CKII) as mentioned in the Methods section. When we compared the intensities of the radioactive signals in the experimental lane (i.e. CIAP- and BAP-treated samples) with those in the control lane, we found that the radioactive signals decreased (Figure 2A
, lanes 58). The Coomassie Brilliant Blue-stained gel (Figure 2B
) indicates the constant amount of P protein used in the experiment. This indicates that the phosphatases used in this experiment are active and the phosphate group(s) incorporated by LAK can be removed by phosphatases. This result suggests that there might be a conformational alteration of the P protein due to CKII-mediated phosphorylation which in turn hides the CKII incorporated phosphate group so that it is no longer accessible to phosphatases used in this experiment. The recombinant P protein could not be phosphorylated by LAK in the presence of heparin whereas under the same conditions the CKII-phosphorylated P protein could be phosphorylated by LAK. This demonstrates the sequential phosphorylation of P protein by CKII and LAK.
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Proteins bind to the Phenyl Sepharose column matrix as a function of exposed hydrophobic surface area (Yamamato, 1991). Phosphorylation induces a small but significant decrease in the binding of P protein to the Phenyl Sepharose column matrix. Most of the phosphorylated P protein can be eluted at a lower concentration of ethylene glycol (Figure 3). This clearly indicates the decrease in the exposed hydrophobic surface area of P protein after phosphorylation. Hence phosphorylation-induced alteration in the conformation changes the accessible hydrophobic surface area.
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Protein fingerprinting can often be used as a very good tool in identifying protein conformational alterations (Calvert and Gratzer, 1978). If the altered three-dimensional structure changes the accessible protease digestion sites, the digestion pattern changes when the proteins are subjected to limited protease digestion. We attempted a similar approach to study the conformational alteration of CHPP protein by limited protease digestion with trypsin and chymotrypsin. Both unphosphorylated and in vitro phosphorylated P proteins were subjected to partial protease digestion with the two proteases.
Partial digestion with trypsin showed changes at two positions as shown in the inset in Figure 4. One peptide migrating around 25 kDa in 16% SDSPAGE appeared in the unphosphorylated samples when digested partially with 2 µg/ml trypsin. This band was absent in the phosphorylated sample at the corresponding protease concentration but appeared at relatively higher concentration of protease. Another peptide migrating just below the previous one appeared when the unphosphorylated sample was digested with 4 µg/ml trypsin, but no new band appeared at this position when the phosphorylated sample was digested with the same amount of protease. The difference in the pattern at these positions remained unaltered when digested with 6 and 8 µg/ml of trypsin. The situation was different in another region. Only one peptide had appeared in the region around 14 kDa in the unphosphorylated sample, whereas there were two peptides migrating closely in the same region in the phosphorylated sample when digested with a larger amount of proteases (68 µg/ml).
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Phosphorylation induces the dimerization of P protein
In order to determine the stoichiometry of multimerization of P protein, the His-tag dilution method (Gao et al., 1996) was applied. It was found that N-terminal His-tagged P protein could support transcription after phosphorylation with CKII (data not shown). It was also found that the elution profile of the phosphorylated His-tagged protein from the gel filtration column (Sephacryl S-300) corresponds to the oligomeric molecule and was different from the unphosphorylated one. Based on these observations, we used the His-tag dilution assay method to determine the stoichiometry of multimerization.
To a constant amount of the tagged protein, increasing amounts of untagged protein were added and phosphorylated in vitro. After recovering the multimer containing the tagged subunit on Ni beads, we analyzed the sample by 10% SDSPAGE. As untagged protein moves faster than the tagged protein, after Coomassie Brilliant Blue staining we could quantify two types of proteins by laser densitometric scanning. Plotting molar ratio against weight ratio, we obtained the saturating molar ratio value of the untagged:tagged protein as 1:1 (Figure 6), indicating the involvement of one tagged and one untagged subunit in the homomultimer, which supports the formation of a PP dimer upon phosphorylation in vitro. The His-tag dilution assay was also performed by mixing His-tag P and the P protein without phosphorylation and it was found that no untagged P protein could be eluted through the Ni-NTA agarose column, demonstrating that unphosphorylated P could not dimerize (data not shown).
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Previously the CKII target site was mapped and it was found that Ser62 is the only site for CKII phosphorylation. To check the phosphorylation status of the truncated P protein, the deletion mutants of CHPP protein were subjected to in vitro phosphorylation by CKII along with the P protein as the control. It was found that the P177 and P47233 proteins were phosphorylated by CKII like the wild-type P protein (Figure 7A and B), whereas neither the P137293 nor the P78293 proteins were phosphorylated under identical conditions. This indicates that CKII is phosphorylating the deletion mutants, which contain Ser62.
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To determine the role of different regions of the P protein in PP homodimerization, we performed a modified His-tag dilution assay. In this case, different truncated P proteins having His-tag at the N-terminal end replaced the His-tagged P protein. After in vitro kinasing, the complexes containing at least one tagged subunit or the unreacted tagged protein were purified using Ni-NTA agarose and analyzed by 10% SDSPAGE. From the results (Figure 8), it is clear that only the deletion mutant P177 protein can form a dimer with the wild-type full-length P protein when phosphorylated in vitro. However, the deletion mutants P47233, P137293 and P78293 failed to form a dimer with the wild-type P protein even after incubation with CKII. This indicates the possibility of involvement of the N-terminal 77 amino acid-containing region in PP dimerization. The in vitro phosphorylated deletion mutant P47230 protein fails to dimerize with the wild-type protein, which rules out the possibility of the involvement of the 4777 amino acid residues in dimer formation. Hence, from the His-tag dilution assay, it is clear that there is no direct involvement of the C-terminal amino acids in PP dimerization and the N-terminal 46 amino acid residues are important for the formation of the dimer.
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Discussion |
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The experiments reported in this paper provide further characterization of the phosphorylation-induced conformational alteration of the P protein of CHP virus by some biochemical techniques. We have also conclusively proved the formation of a homodimer of P protein of CHP virus due to phosphorylation. In a previous paper we demonstrated that phosphorylation of P protein by CKII is essential for transcriptional activity (Chattopadhyay et al., 1997).
The stability of the phosphate groups of P protein deserves special attention. The first biochemical approach to monitoring the change in the tertiary structure of P protein was the inaccessibility of the CKII-incorporated phosphate group to phosphatases whereas the phosphate group(s) incorporated by LAK can be dephosphorylated by the same phosphatases. We have seen that no phosphorylation of recombinant P protein occurs by LAK in the presence of heparin, a CKII inhibitor (data not shown). Hence phosphorylation by LAK is possible only if P protein is phosphorylated first with CKII. This proves that after phosphorylation by CKII the protein undergoes a change in conformation which then becomes the substrate for another step of phosphorylation by LAK. Although the precise role of second phosphorylation in CHP virus is still unknown, it is clear that the change in the three-dimensional structure of P protein after phosphorylation by CKII alters the structure of P protein in such a way that the phosphate group becomes buried within the core of the protein moiety and thus becomes resistant to phosphatases, whereas in the case of second phosphorylation by LAK the incorporated phospate groups become phosphatase sensitive. The decrease in the binding affinity of the phosphorylated protein to Phenyl Sepharose gives an idea of the decreased hydrophobic surface area upon phosphorylation. Ethylene glycol affects the binding of P protein to Phenyl Sepharose much less in the unphosphorylated form than in the phosphorylated form. All these results together may indicate that phosphorylation induces a change from an `open' to `closed' conformation of the protein.
The partial protease digestion patterns demonstrate clearly that there is a change in the tertiary structure and this was reflected in the availability of digestion sites of the proteases. However, it is also noticeable that the overall digestion pattern of the P protein changes only at two or three places although the changes are distinct. Although it is not certain that the peptides migrating at the same position in the case of both the unphosphorylated and phosphorylated proteins are the same peptide, it is conceivable that the changes occurred at the region most probably surrounding the sites of phosphorylation as indicated by phosphatase resistance. However, it does not exclude the possibility that the introduced phosphate group is buried in the monomermonomer interface in the PP homodimer and in turn is not accessible to phosphatases and to proteases. The partial protease digestion pattern of unphosphorylated and phosphorylated CHPP protein also indicates a domain structure of CHPP protein.
Gel filtration through a Sephacryl S-300 column indicates the possibility of dimerization of unphosphorylated monomeric P protein (Chattopadhyay et al., 1997). Since this type of chromatography does not measure the molecular weight, molecules are separated from each other on these columns based on their shape and size. We can only have an idea of the Stokes radius of the molecule. For instance, if the molecule were a highly `coiled blob' before phosphorylation and then became an `extended rod' after phosphorylation, the elution profile may follow the profile obtained in the size exclusion chromatographic results. However, by the His-tag dilution assay it was conclusively proved that phosphorylation induces dimerization which is most probably needed for the transcription activation property of the P protein.
In the case of the P protein of VSV (IND), it was found that the N-terminal 130 amino acids are involved in oligomerization (Gao and Lenard, 1995a,b
). The computer prediction of the five rhabdovirus P proteins available, VSV-IND, VSV-NJ, rabies, CHP and Piry, indicates that except for Piry virus, all P proteins contain a putative helix of ~30 residues near the N-termini with an elevated coiled-coil potential. In the case of paramyxovirus, a `coiled-coil' region at the C-terminal end was found to be responsible for oligomerization (Curran et al., 1995
). In CHP virus it was reported that phosphorylation induces an `unstructured' to `structured' transition in the N-terminal region (residues 4969) of P protein surrounding the CKII target site (Raha et al., 1999
). As evidenced by the His-tag dilution assay, the direct involvement of the N-terminal 46 amino acid residues in PP dimerization also indicates the role of the domain near the CKII target region, which was previously shown to be responsible for transcription activation (Chattopadhyay et al., 1997
). The CKII target region is predicted to be disordered, and upon phosphorylation undergoes a definite change in the tertiary structure that may help the N-terminal coiled-coil region of the P protein to form the dimer. The results reconcile fairly well with the idea that CKII may act as an `architectural protein' to sculpt the P protein into the precise three-dimensional shape which helps in activation of transcription by the virion RNA polymerase. However, only detailed NMR and crystallographic studies can pinpoint the CKII-mediated dynamic changes at the N-terminal region of the P protein upon phosphoryation to confer the role of phosphoprotein in the viral life cycle.
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Notes |
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3 Present address: Department of Molecular Biology, Cleveland Clinic Foundation, Cleveland, Ohio, USA
4 To whom correspondence should be addressed. E-mail: dhruba{at}cubmb.ernet.in
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Acknowledgments |
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References |
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Received September 21, 1999; revised February 4, 2000; accepted April 4, 2000.