Virulence Factor of Potato Virus Y, Genome-attached Terminal Protein VPg, Is a Highly Disordered Protein
2007; Elsevier BV; Volume: 283; Issue: 1 Linguagem: Inglês
10.1074/jbc.m705666200
ISSN1083-351X
AutoresRenata Grzela, Ewa Szołajska, Christine Ebel, Dominique Madern, Adrien Favier, Izabela Wojtal, Włodzimierz Zagórski, Jadwiga Chroboczek,
Tópico(s)Bacteriophages and microbial interactions
ResumoPotato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5′ end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host. Potato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5′ end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host. It is generally accepted that a protein should attain stable folded conformation to perform its specific physiological functions. However, we are beginning to realize that many proteins contain large unfolded regions, and there are examples of entire proteins lacking regular secondary and tertiary structures (1Romero P. Obradowic Z. Kissinger C.R. Villafranca J.E. Garner E. Guilliot S. Dunker A.K. Pac. Symp. Biocomput. 1998; : 437-448PubMed Google Scholar). There is abundant evidence that the unstructured state, common to all living organisms, is essential for basic cellular functions. Approximately 10% of proteins are predicted to be fully disordered (2Tompa P. Trends Biochem. Sci. 2002; 27: 527-533Abstract Full Text Full Text PDF PubMed Scopus (1677) Google Scholar). Proteins should fulfill multiple functions through interactions with distinct partners, which requires structured proteins to use separate binding surfaces or domains. In contrast, intrinsically unstructured proteins can, because of their malleability, use the same regions or overlapping surfaces for different interactions by taking advantage of their capacity to adopt different conformations upon binding. Potyvirus VPg is a virus-coded terminal protein of ∼22 kDa, attached to the 5′ end of virus RNA genome. During the virus life cycle VPg is expressed as a part of a larger polyprotein, which self-processes into mature viral protein components. VPg is the N-terminal part of one of the first products, VPg-Pro (also called NIa), liberated by polyprotein proteolysis. At the end of the viral cycle VPg is attached to the 5′ terminus of the progeny genome and is packaged into virions along with viral RNA. VPg is required for potyvirus infectivity. The latter is abolished when genomic PVY 3The abbreviations used are:PVYpotato virus YTFE2,2,2-trifluoroethanolDTTdithiothreitolGSTglutathione S-transferaseeIFeukaryotic initiation factor RNA is treated with proteinase K (3Herbert T.P. Brierley I. Brown T.D. J. Gen. Virol. 1997; 78: 1033-1040Crossref PubMed Scopus (114) Google Scholar). Moreover, mutation of the tyrosine residue involved in the linkage between RNA and the VPg protein is lethal for virus growth and replication (4Murphy J.F. Klein P.G. Hunt A.G. Shaw J.G. Virology. 1996; 220: 535-538Crossref PubMed Scopus (83) Google Scholar, 5Mitra T. Sosnovtsev S.V. Green K.Y. J. Virol. 2004; 78: 4931-4935Crossref PubMed Scopus (36) Google Scholar). VPg seems to be involved in multiple interactions, with both other viral products and host proteins. It has been implicated in translation (6Lellis A.D. Kasschau K.D. Whitham S.A. Carrington J.C. Curr. Biol. 2002; 12: 1046-1051Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar), long distance movement in plant tissue (7Schaad M.C. Lellis A.D. Carrington J.C. J. Virol. 1997; 11: 8624-8631Crossref Google Scholar), and replication (8Fellers J. Wan J. Hong Y. Collins G.B. Hunt A.G. J. Gen. Virol. 1998; 79: 2043-2049Crossref PubMed Scopus (60) Google Scholar). It has also been shown to interact with viral RNA polymerase, suggesting a role, possibly as a primer, in viral RNA synthesis (8Fellers J. Wan J. Hong Y. Collins G.B. Hunt A.G. J. Gen. Virol. 1998; 79: 2043-2049Crossref PubMed Scopus (60) Google Scholar, 9Hong Y. Levay K. Murphy J.F. Klein P.G. Shaw J.G. Hunt A.G. Virology. 1995; 214: 159-166Crossref PubMed Scopus (78) Google Scholar, 10Puustinen P. Makinen K. J. Biol. Chem. 2004; 279: 38103-38110Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). VPg, as part of the VPg-Pro protein, has been observed to translocate to the host cell nucleus at the beginning of infection, possibly by an import independent mechanism (11Restrepo M.A. Freed D.D. Carrington J.C. Plant Cell. 1990; 2: 987-998Crossref PubMed Scopus (299) Google Scholar, 12Carrington J.C. Freed D.D. Leinicke A.J. Plant Cell. 1991; 3: 953-962PubMed Google Scholar, 13Schaad M.C. Haldeman-Cahill R. Cronin S. Carrington J.C. J. Virol. 1996; 70: 7039-7048Crossref PubMed Google Scholar, 14Beauchemin C. Boutet N. Laliberte J.F. J. Virol. 2007; 81: 775-782Crossref PubMed Scopus (132) Google Scholar). Indeed, VPg contains a two-partite nuclear localization signal (amino acid residues 1–11 and 43–57 in VPg of tobacco etch virus potyvirus (12Carrington J.C. Freed D.D. Leinicke A.J. Plant Cell. 1991; 3: 953-962PubMed Google Scholar)). However, the role of VPg in the nucleus is not clear because mature potyvirus particles accumulate in the cytoplasm of infected plants. Nevertheless, knocking out VPg nuclear translocation inhibits genome amplification, suggesting that the nuclear localization signal might be in a region critical for RNA replication (13Schaad M.C. Haldeman-Cahill R. Cronin S. Carrington J.C. J. Virol. 1996; 70: 7039-7048Crossref PubMed Google Scholar). Functional VPg seems to be complexed with another viral product, protein 6kDa, which may mediate VPg binding to membranes at sites of RNA replication (15Restrepo-Hartwig M.A. Carrington J.C. J. Virol. 1994; 68: 2388-2397Crossref PubMed Google Scholar, 16Léonard S. Viel C. Beauchemin C. Daigneault N. Fortin M.G. Laliberte J.F. J. Gen. Virol. 2004; 85: 1055-1063Crossref PubMed Scopus (121) Google Scholar). potato virus Y 2,2,2-trifluoroethanol dithiothreitol glutathione S-transferase eukaryotic initiation factor Several studies have reported on the interaction between the potyvirus VPg protein and the eukaryotic translation initiation factor eIF4E, which is the mRNA 5′ cap-binding protein and in plants occurs in two isoforms (17Wittmann S. Chatel H. Fortin M.G. Laliberte J.F. Virology. 1997; 234: 84-92Crossref PubMed Scopus (220) Google Scholar, 18Léonard S. Plante D. Wittmann S. Daigneault N. Fortin M.G. Laliberté J.F. J. Virol. 2000; 74: 7730-7737Crossref PubMed Scopus (244) Google Scholar, 19Duprat A. Caranta C. Revers F. Menand B. Browning K.S. Robaglia C. Plant J. 2002; 3: 927-934Crossref Scopus (208) Google Scholar, 20Kang B.C. Yeam I. Frantz J.D. Murphy J.F. Jahn M.M. Plant J. 2005; 42: 392-405Crossref PubMed Scopus (187) Google Scholar, 21Grzela R. Strokovska L. Andrieu J.P. Dublet B. Zagorski W. Chroboczek J. Biochimie (Paris). 2006; 88: 887-896Crossref PubMed Scopus (38) Google Scholar). This interaction seems to be crucial for a productive virus cycle. Natural plant resistance to potyvirus infection has been shown to stem from the inability of VPg to interact with eIF4E, as a result of amino acid mutations either in VPg (22Borgstrom B. Johansen I.E. Mol. Plant Microbe Interact. 2001; 14: 707-714Crossref PubMed Scopus (53) Google Scholar) or in eIF4E (19Duprat A. Caranta C. Revers F. Menand B. Browning K.S. Robaglia C. Plant J. 2002; 3: 927-934Crossref Scopus (208) Google Scholar, 23Ruffel S. Dussault M.H. Palloix A. Moury B. Bendahmane A. Robaglia C. Caranta C. Plant J. 2002; 32: 1067-1075Crossref PubMed Scopus (281) Google Scholar). VPg of turnip mosaic virus also interacts with the host poly(A)-binding protein (16Léonard S. Viel C. Beauchemin C. Daigneault N. Fortin M.G. Laliberte J.F. J. Gen. Virol. 2004; 85: 1055-1063Crossref PubMed Scopus (121) Google Scholar). Furthermore, turnip mosaic virus VPg has been shown to interact with a plant protein called potyvirus VPg-interacting protein through the 16 amino acids at the VPg N terminus (24Dunoyer P. Thomas C. Harrison S. Revers F. Maule A. J. Virol. 2004; 78: 2301-2309Crossref PubMed Scopus (105) Google Scholar). Reduced expression of potyvirus VPg-interacting protein has been observed to diminish susceptibility to turnip mosaic virus infection, whereas eliminating the interaction with VPg resulted in reduced virus cell-to-cell and systemic movement (24Dunoyer P. Thomas C. Harrison S. Revers F. Maule A. J. Virol. 2004; 78: 2301-2309Crossref PubMed Scopus (105) Google Scholar). Finally, it has been shown that VPg of infecting PVY can be phosphorylated by plant kinases (25Ivanov K.I. Puustinen P. Merits A. Saarma M. Makinen K. J. Biol. Chem. 2001; 276: 13530-13540Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Consequently it has been hypothesized that VPg phosphorylation could trigger disassembly of infecting virions and subsequent initiation of potyvirus protein synthesis in infected cells (26Puustinen P. Rajamaki M.L. Ivanov K.I. Valkonen J.P. Makinen K. J. Virol. 2002; 76: 12703-12711Crossref PubMed Scopus (55) Google Scholar). All of these data show first that VPg is potyvirus virulence factor and second that VPg is a multifunctional protein. We demonstrate here that the structure of VPg of PVY is characteristic of an intrinsically disordered protein, which may help to explain the multiple functions played by this protein in the potyvirus life cycle. Proteins—Recombinant PVY VPg (accession number Z29526, potato virus Y strain 0, British isolate) and wheat iso isoform of eIF4E were expressed and purified as described (21Grzela R. Strokovska L. Andrieu J.P. Dublet B. Zagorski W. Chroboczek J. Biochimie (Paris). 2006; 88: 887-896Crossref PubMed Scopus (38) Google Scholar). Both plant eIF4E isoforms are able to interact with PVY VPg; however, the iso isoform interacts more tightly (21Grzela R. Strokovska L. Andrieu J.P. Dublet B. Zagorski W. Chroboczek J. Biochimie (Paris). 2006; 88: 887-896Crossref PubMed Scopus (38) Google Scholar). Polyacrylamide Gel Analysis—The analysis was carried out under denaturing conditions according to Laemmli (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar) or under semi-denaturing conditions, that is, with samples suspended without boiling in Laemmli sample buffer devoid of SDS and β-mercaptoethanol. Proteolytic Analysis—VPg (15 μg) was incubated with chymotrypsin (2.5, 12.5, and 60 ng) for 10 min at room temperature in 25 μl of 50 mm phosphate buffer, pH 7.0, containing 300 mm NaCl. The reactions were stopped with the addition of Laemmli sample buffer, and the proteins were analyzed using 17% SDS-PAGE. For N-terminal amino acid analysis, proteolytic fragments were transferred onto a Problot (ABI) membrane and sequenced as described (21Grzela R. Strokovska L. Andrieu J.P. Dublet B. Zagorski W. Chroboczek J. Biochimie (Paris). 2006; 88: 887-896Crossref PubMed Scopus (38) Google Scholar). Disorder Prediction—The VPg folding predictions were carried out using two software packages: PONDR VL-XT, XL1_XT and CAN_XT, which predict natural disordered regions (www.pondr.com), and FoldIndex©, which predicts to what extent a given protein sequence is intrinsically unfolded (28Prilusky J. Zeev-Ben-Mordehai T. Rydberg E. Felder C. Silman I. Sussman J.L. Bioinformatics. 2005; 21: 3435-3438Crossref PubMed Scopus (805) Google Scholar). FoldIndex© can be used as a Web Service for remote and automatic data processing by accessing this URL: bioportal.weizmann.ac.il/fldbin/findex?m=xml&sq=SEQUENCE. The analyses have been performed using default values. Dynamic Light Scattering—Protein samples were dialyzed against prefiltered (0.22-μm Millipore filters) 50 mm Hepes buffer, pH 7.6, containing 200 mm KCl, 2 mm DTT, 0.5 mm EDTA, and 10% glycerol. The samples were placed in a reduced volume cuvette (45 μl; Greiner). The automated measurements were collected with a Zetasizer Nano ZS instrument, using a 2 °C incremental temperature ramp and a 2-min equilibrium delay at each temperature. The data were adjusted using the cumulants method. Analytical Ultracentrifugation—Sedimentation velocity experiments were performed at 42,000 rpm and 10 °C, using an AN-60 rotor of Beckman XL-I analytical ultracentrifuge and two-channel centerpiece with 1.2-cm path length. Three different VPg preparations were analyzed: VPg purified in the presence (5 mm) or absence of DTT, and VPg was stored for 2 weeks at 4 °C without DTT. Each preparation contained ∼0.15 mg of protein/ml (A280 nm = 0.1) in 20 mm Tris-HCl buffer, pH 7.0, containing 300 mm NaCl. The scans were recorded overnight every 6 min at 280 nm using a 0.003-cm radial spacing. The data for each preparation were analyzed as a continuous distribution c(s) of sedimentation coefficients, s, with the program Sedfit. The program Sedphat was used to perform a global analysis of the three sets of data (all together) in terms of a mixture of noninteracting VPg species with fixed molar masses of the monomer and dimer. These programs are available free at www.analyticalultracentrifugation.com (29Schuck P. Biophys J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3065) Google Scholar) (for a recent review of analytical ultracentrifugation data treatment, see Ref. 30Ebel C. Protein Structures: Methods in Protein Structure and Stability Analysis. Nova Science Publishers, Inc., Hauppauge, NY2007Google Scholar). Corrected s20,w were obtained in the usual way using a partial specific volume of v̄ = 0.742 ml/g estimated from amino acid composition with the program Sednterp (www.jphilo.mailway.com), using the experimental solvent density, ρ = 1.013 g/ml, and viscosity η = 1.35 mPa/s. The Svedberg equation relates s to protein molar mass M, and hydrodynamic radius Rh as follows, s=M(1-ν¯ρ/(NA6πηRh))(Eq. 1) where NA is Avogadro's number. Theoretical values for a globular compact monomer and dimer were calculated with Rh = f/fmin × Rmin, using the frictional ratio f/fmin = 1.25. Rmin is the minimum theoretical value for Rh, corresponding to the anhydrous volume v̄M/NA = (4/3)π R3min. Far-UV CD—CD spectra of VPg at 25 °C were recorded using a Jobin Yvon CD6 spectropolarimeter. The wavelength range used was 180–260 nm, with a 1-nm interval and an integration time of 4 s in continuous scanning mode. The samples were contained in 0.1-cm optical path length quartz cuvettes. VPg (0.2 mg/ml) was in 50 mm sodium phosphate, pH 7.0, containing 60 mm NaCl with or without 20% TFE. To monitor VPg thermal stability CD spectra were recorded after 30 min of incubation at various temperatures up to 95 °C. NMR Spectroscopy—Conventional 1H one-dimensional NMR using excitation sculpting for water suppression purposes (31Hwang T.L. Shaka A.J. J. Magn. Reson. A. 1995; 112: 275-279Crossref Scopus (1565) Google Scholar) and one-dimensional HET-SOFAST experiments (32Schanda P. Forge V. Brutscher B. Magn. Reson. Chem. 2006; 44: S177-S184Crossref PubMed Scopus (60) Google Scholar) were performed on a Varian DirectDrive 600 spectrometer equipped with a triple resonance cold-probe. The one-dimensional HET-SOFAST experiments are composed of two data sets recorded with and without a band-selective inversion pulse covering the aliphatic region. The data were acquired at 10 or 30 °C. Data processing was done using the VnmrJ software (Varian, Inc.). The time domain data were multiplied with a 90° phase-shifted sine-bell apodization function and zero-filled to 8192 complex data points prior to Fourier transformation. The reference and saturated intensities were obtained by integrating the spectra from ∼7.0 to 10.0 ppm. Structure Prediction and Proteolysis—Full-length PVY polyprotein was analyzed using FoldIndex©. This program furnishes an estimate of the degree of protein disorder. It was developed from the algorithm proposed by Uversky et al. (36Uversky V.N. Gillespie J.R. Fink A.L. Proteins. 2000; 41: 415-427Crossref PubMed Scopus (1773) Google Scholar) and is based on the mean net charge and hydrophobicity of the polypeptide chain. The results clearly show that most of VPg, which is located between amino acids 336 and 523 of PVY polyprotein, lies in the unfolded region of the polyprotein, in contrast to other PVY proteins: CI, 6K2, and NI-Pro (the C-terminal part of NIa proteins containing the protease domain only) (Fig. 1A). The structure of VPg was also probed experimentally with proteases, and the results were compared with the PONDR disorder prediction. There are 31 consensus cleavage sites for trypsin alone, distributed throughout the VPg amino acid sequence. However, when treated with trypsin or chymotrypsin, VPg was cleaved mainly between residues 11–21 and 41–61 (Fig. 1B, lower panel). Thus, the VPg N-terminal region is predominantly accessible to proteases. We infer that also the C-terminal part of the VPg is cleaved as the N-terminal analysis revealed the presence of peptides with the same N termini but with a different mobility on SDS-PAGE (Fig. 1B, upper panel). C-terminal short peptides could not be identified because they ran out of the gel. The results of proteolytic analysis agree with PONDR predictions. The latter unambiguously indicates that the regions between residues 62–83, 118–130, and 140–149 are ordered and suggests that the N-terminal region as well as fragments delineated by residues 84–117, 130–139, and 150–188 in the C-terminal region are disordered (Fig. 1C). Effect of Temperature on VPg—Structured proteins are generally denatured by heat. Indeed, eIF4E aggregates completely, when maintained at 100 °C for 15 min, implying denaturation, whereas VPg endures this treatment without precipitating (Fig. 2A). Assuming that GST would have a protective effect, we applied the same treatment to the fusion protein GST-eIF4E, which, however aggregated, as did bovine serum albumin (results not shown). The dynamic light scattering technique makes it possible to monitor unfolding or denaturation caused by changes in pH or temperature. The protein melting point temperature Tm obtained from dynamic light scattering analysis is indicative of thermal stability. In the absence of aggregation-inhibiting agents, interpolymer hydrophobic interactions can quickly lead to nonspecific aggregation of the denatured polypeptide chains. The change in mean particle size that accompanies protein denaturation was measured using a 12–65 °C thermal gradient. In this temperature range the VPg preparation showed no signs of denaturation (Fig. 2B). Parallel measurements done with eIF4E produced a typical denaturation profile with a slow increase in particle size beginning at ∼55 °C, followed by a rapid increase at 60 °C. VPg Oligomerization—VPg contains one cysteine (Cys150) among its 188 amino acid residues. When VPg was prepared under nonreducing conditions and run on semi-denaturing SDS-PAGE, a slowly migrating band of 44 kDa, consistent with the molecular weight calculated for VPg dimer (21Grzela R. Strokovska L. Andrieu J.P. Dublet B. Zagorski W. Chroboczek J. Biochimie (Paris). 2006; 88: 887-896Crossref PubMed Scopus (38) Google Scholar) was observed in addition to a major band (22kDa) corresponding to the monomeric protein (Fig. 3A, lane 1). The same preparation run on denaturing PAGE showed only one band migrating at 22 kDa (Fig. 3A, lane 3). The amount of the 44-kDa form increased with time of storage at 4 °C but disappeared when VPg was incubated with 5 mm DTT (Fig. 3A, lane 2). When N-ethylmaleimide was added at the beginning of VPg purification, the protein appeared in a monomeric form only (Fig. 3B, lanes 1 and 2). N-Ethylmaleimide forms covalent bonds with sulfhydryl groups of cysteines and prevents S-S bond formation. Similar gel analysis performed on VPg C-terminally truncated at residue 139 showed only one band migrating with the monomer molecular weight under nonreducing conditions (Fig. 3B, lanes 3 and 4). Moreover, no VPg dimers were observed in freshly expressing insect cells (Fig. 3B, lane 6; His-tagged VPg is somewhat retarded in comparison with untagged protein). It seems thus that the VPg dimer arises during VPg purification or manipulation, when atmospheric oxygen induces formation of disulfide bonds. It is worth noting that the RNAs prepared from three different potyviruses were found in an apparently VPg-dependent aggregated form (37Luciano C.S. Murphy J.F. Rhoads R.E. Shaw J.G. J. Gen. Virol. 1991; 72: 205-208Crossref PubMed Scopus (7) Google Scholar). The propensity for oligomerization has been observed previously for VPg proteins of clover yellow vein virus (38Yambao M.L. Masuta C. Nakahara K. Uyeda I. J. Gen. Virol. 2003; 84: 2861-2869Crossref PubMed Scopus (47) Google Scholar) and turnip mosaic virus (14Beauchemin C. Boutet N. Laliberte J.F. J. Virol. 2007; 81: 775-782Crossref PubMed Scopus (132) Google Scholar). The appearance of dimers caused by the formation of disulfide bridges might have changed or impaired VPg functionality. For example, stabilization of Agrobacterium tumefaciens VirB7, an outer membrane-associated lipoprotein, is correlated with its ability to form disulfide cross-linked homodimers (39Spudich G.M. Fernandez D. Zhou X.R. Christie P.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7512-7517Crossref PubMed Scopus (96) Google Scholar). Similarly, cysteine oxidation prevents p53 dimerization and hence inhibits DNA binding (40Delphin C. Cahen P. Lawrence J.J. Baudier J. Eur. J. Biochem. 1994; 223: 683-692Crossref PubMed Scopus (72) Google Scholar). In some redox-responsive proteins, the formation of disulfide bonds modulates protein-protein interactions; upon oxidation Escherichia coli chaperone Hsp33 undergoes a large conformational transition to a state that can bind polypeptide substrates and rescue them from aggregation (41Graumann J. Lilie H. Tang X. Tucker K.A. Hoffman J.H. Vijayalakshmi J. Saper M. Bardwell J.C. Jakob U. Structure. 2001; 9: 377-387Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). To explore the effect of VPg dimerization on function through a change in interaction with its host partner, eIF4E, we studied the complex formation using monomeric and dimeric VPg in parallel. As shown in Fig. 3C (lanes E), no difference is observed in the interaction of DTT-treated VPg (monomeric form) and stored VPg (dimeric form) with GST-eIF4E immobilized on a GST column. This suggests that VPg utilizes for dimerization regions different from those implicated in its interaction with eIF4E. Note that the N-terminal part of VPg seems to interact with host eIF4E (18Léonard S. Plante D. Wittmann S. Daigneault N. Fortin M.G. Laliberté J.F. J. Virol. 2000; 74: 7730-7737Crossref PubMed Scopus (244) Google Scholar, 21Grzela R. Strokovska L. Andrieu J.P. Dublet B. Zagorski W. Chroboczek J. Biochimie (Paris). 2006; 88: 887-896Crossref PubMed Scopus (38) Google Scholar), whereas the C-terminal part is involved in dimerization (Fig. 3B, lanes 3 and 4). Also oligomerization of clover yellow vein virus VPg involves the C-terminal part of the protein (38Yambao M.L. Masuta C. Nakahara K. Uyeda I. J. Gen. Virol. 2003; 84: 2861-2869Crossref PubMed Scopus (47) Google Scholar). Analytical Ultracentrifugation—Sedimentation velocity experiments were performed to evaluate the shape and oligomeric state of VPg under nondenaturating conditions. As mentioned earlier, VPg has a tendency to oligomerize upon prolonged storage. From c(s) analysis (Fig. 4), VPg stored at –20 °C and thawed (VPgM+D sample) has a maximum mean s20,w of 2.5 S. VPg stored for 2 weeks at 4 °C (VPgD sample) and believed to be a dimer has a larger mean s20,w value of 3.0 S, whereas VPg purified in the presence of DTT (monomer, VPgM) has the smallest s20,w mean value of 2.0 S. The three sets of data were also modeled as a mixture of noninteracting monomer and dimer species. In this approach, the molar mass is fixed, whereas the sedimentation coefficient for monomer and dimer, as well as the proportion of both species in different samples, are fitted. The results are compatible with the presence of ∼90% monomer in the VPgM sample, ∼90% dimer in the VPgD sample, and equal amounts of the monomer and dimer in the VPgM+D sample (Tables 1 and 2). The quality of our data (not shown) does not allow us to distinguish between rapid equilibrium (as suggested by the intermediate position of the VPgM+D peak in Fig. 4) and slow equilibrium (corresponding to an analysis with two species). The results of the monomer/dimer mixture analysis gives the s20,w values 1.7 and 3.0 S for VPg monomer and dimer, respectively. These values are rather small compared with those calculated for globular compact species, namely 2.1 and 3.4 S (Table 2). The results thus imply an elongated structure for both monomeric and dimeric VPg.TABLE 1Percentage of monomer and dimer in each sampleSamplesMonomerDimer%%VPgM8911VPgM+D4753VPgD892 Open table in a new tab TABLE 2Global analysis of sedimentation velocity of the three VPg samples in terms of two noninteracting species: monomer and dimer (Sedphat program)Molecular massFitted S20wTheoretical S20w with f/fmin = 1.25kDaSSMonomer22.21.72.1Dimer44.43.03.4 Open table in a new tab Circular Dichroism—Intrinsically unstructured proteins possess distinctive far-UV CD spectra with a characteristic deep minima in the vicinity of 200 nm and relatively low ellipticity at 215 and 222 nm resulting from the low content of ordered secondary structure. The CD spectra of monomeric and dimeric VPg (Fig. 5A) are identical and exhibit negative ellipticity near 205 nm, a shoulder at 220 nm, and a weak positive maximum of ∼190 nm. These spectral features have been assigned to a group of β-sheet-rich proteins containing short segments of β-strands (so called beta-II proteins) (42Sreerama N. Woody R.W. Protein Sci. 2003; 12: 384-388Crossref PubMed Scopus (163) Google Scholar). The VPg spectrum was not changed by the presence of 10% TFE but was significantly modified by 20% TFE (Fig. 5A, dots). This spectrum has a positive molar ellipticity, centered at ∼200 nm and a negative band at 228 nm, strikingly similar to spectra observed with beta-I rich proteins (42Sreerama N. Woody R.W. Protein Sci. 2003; 12: 384-388Crossref PubMed Scopus (163) Google Scholar). The effect of temperature was tested (Fig. 5B), and in common with other unfolded proteins (43Sanchez-Puig N. Veprintsev D.B. Fersht A.R. Protein Sci. 2005; 14: 1410-1418Crossref PubMed Scopus (56) Google Scholar), no cooperative transition of the molar ellipticity was observed. Our data show that VPg does not have a compact structure, nor does it contain significant secondary elements. In addition, it appears that VPg undergoes a structural reorganization in TFE, which is known to favor local interactions by increasing the propensity for helix formation. Because the dielectric constant of TFE is approximately one-third of that of water, charge-mediated interactions should be stronger in TFE; in particular intramolecular hydrogen bonds should be strengthened by the addition of TFE to an aqueous solution (Ref. 44Thomas P.D. Dill K.A. Protein Sci. 1993; 2: 2050-2065Crossref PubMed Scopus (266) Google Scholar and the references therein). NMR Spectroscopy—The degree of dispersion of the proton signals observed by one-dimensional NMR reflects the extent of protein folding. Recently, a new NMR experimental procedure, called HET-SOFAST, has been proposed by Schanda et al. (32Schanda P. Forge V. Brutscher B. Magn. Reson. Chem. 2006; 44: S177-S184Crossref PubMed Scopus (60) Google Scholar), which allows the extent of protein compactness to be quantified using the ratio, λNOE (measure of average proton density), between a reference and a saturated spectrum. Although for compact, well folded prote
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