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A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia
Correspondence
Natalia O. Kalinina
kalinina{at}genebee.msu.ru
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Some plant virus genera belonging to the ‘alpha-like’ supergroup of RNA viruses encode a distinct transport gene module, the triple gene block (TGB). The three partially overlapping TGB genes code for a conserved set of MPs referred to as TGBp1, TGBp2 and TGBp3 (Solovyev et al., 1996; Morozov & Solovyev, 2003). The two smaller proteins (TGBp2 and TGBp3) possess hydrophobic segments and bind to intracellular membrane compartments (Morozov & Solovyev, 2003; Zamyatnin et al., 2004; Haupt et al., 2005; Verchot-Lubicz, 2005; Schepetilnikov et al., 2005). The largest TGB protein, TGBp1, contains an NTPase/helicase sequence domain (Morozov et al., 1989; Koonin & Dolja, 1993). Two types of TGBp1 proteins are distinguished: in the potex-like TGBp1 encoded by filamentous viruses of the Potexviridae, the NTPase/helicase domain comprises the entire TGBp1 sequence, whereas the hordei-like TGBp1 (rod-shaped viruses of the genera Hordeivirus, Benyvirus, Pomovirus and Pecluvirus) also includes an N-terminal extension domain (Morozov & Solovyev, 2003).
In accordance with predictions based on sequence analysis, the protein region comprising the NTPase/helicase domain in both potex-like and hordei-like TGBp1s exhibits NTPase activity and RNA helicase activity in vitro (Rouleau et al., 1994; Donald et al., 1997; Kalinina et al., 1996, 2002; Liou et al., 2000). The TGBp1 NTPase/helicase sequence domain is closely related to replicative helicases of ‘alpha-like’ viruses and belongs to helicases of the superfamily 1 (SF1) (Gorbalenya et al., 1989; Gorbalenya & Koonin, 1993; Koonin & Dolja, 1993). This domain has seven typical sequence motifs, of which motif I and motif II correspond to the ‘Walker A’ and ‘Walker B’ sites found in numerous ATP-binding proteins (Gorbalenya & Koonin, 1993; Leipe et al., 2002). Sequence comparisons of TGBp1 with the related DNA helicases of SF1, for which the X-ray structure has been resolved (Caruthers & McKay, 2002), reveal that TGBp1 contains only two of the four structural domains found in SF1 DNA helicases (Kalinina et al., 2002; Morozov & Solovyev, 2003).
Experimental data suggest that ATPase activity is required for cell-to-cell movement, particularly for plasmodesmata dilation by the potex-like TGBp1 (Angell et al., 1996; Lough et al., 1998, 2000; Malcuit et al., 1999; Morozov et al., 1999; Yang et al., 2000; Howard et al., 2004). The TGBp1 helicase activity is involved in protein/RNA translocation through plasmodesmata to the adjacent cell for both types of viruses with TGB (Morozov & Solovyev, 2003).
TGBp1 of Potato virus X (PVX) has also been shown to be a suppressor of RNA silencing (Voinnet et al., 2000). Recently, random mutation analysis of PVX TGBp1 suggested that silencing suppression is essential for the protein to mediate viral cell-to-cell movement (Bayne et al., 2005). TGBp1 was concluded to possess at least two functions required for viral cell-to-cell movement: one is related to the ability of the protein to suppress RNA silencing, while another is responsible for movement per se. The ATPase activity of TGBp1 might be required for suppression of silencing.
In this study, we have further investigated TGBp1 functional regions. We have shown that the N-terminal part of the TGBp1 proteins of both virus types (potex-like and hordei-like) including three NTPase/helicase motifs, I, Ia and II, and a small sequence upstream of motif I (a compact region of about 100 amino acid residues) is sufficient for ATPase and cooperative RNA-binding activities. Additionally, the functional activities of a conserved positively charged residue upstream of motif I in the NTPase/helicase domain were analysed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) as a template. The 3' primer contained an XbaI site (in italics). The PCR product was digested with EcoRI/XbaI and ligated into vector pQE30 (6x His-tagged expression vector; Qiagen) cut by EcoRI/XbaI. A mutant containing the C-terminal part of TGBp1 of Poa semilatent virus (PSLV) (63K protein) with motifs III–VI deleted (C63KI–II) was obtained similarly: the PSLV C63K coding region was amplified with primers 5'-TTGTGAGCGGATAACAATTTC-3' and 5'-GCTCTAGATTACAAGAGAATTTCAGCGCTTTC-3' using pQE-C63K (Kalinina et al., 2001) as a template. The PCR product was cloned in the pQE31 vector (Qiagen) using EcoRI/XbaI sites. 25K clones containing point mutations (25KR/A and 25KR/AI–II) were obtained by PCR amplification with the primers 5'-ATACCATGGATATTCTCATCAGTAGTTTGAAAAGTTTAGGTTATTCTGCGACTTCC-3' and either 5'-AGCGTCTAGACTATGGCCCTGCGCGGACA-3' with pQE-25K or 5'-CGCTCTAGATTAGTTAGAGTTCCTTGTGGTG-3' with pQE-25KI–II as templates. The resulting PCR products were cut with NcoI (restriction site shown in bold) and XbaI (in italics) and then ligated into a modified pQE31 vector, where an NcoI site was inserted in the polylinker. To obtain this plasmid, the pQE31 vector was fully digested with BamHI/HindIII, removing the original polylinker. Two partially overlapping primers with a new NcoI site (shown in bold) (5'-GATCCCGGGCCCATGGA-3' and 5'-AGCTTCCATGGGCCCGG-3') were then annealed and ligated into the vector instead of the original polylinker. Two constructs were produced using similar procedures: pQE-25KR/A and pQE-25KR/AI–II.
Point mutations in the C63K protein (mutants C63KK/A and C63KK/AI–II) were introduced by PCR amplification of pQE-C63K with two sets of primers, 5'-TTGTGAGCGGATAACAATTTC-3'/5'-ACGGGCCCTCAGTTGATTTCTTGGTCG-3' (ApaI site in bold) and 5'-GAGGGCCCGTGGTTACTCGTTAGGAAC-3'/5'-GCTCTAGATTACAAGAGAATTTCAGCGCTTTC-3' (XbaI site in italics), respectively. To obtain the construct pQE-C63KK/AI–II, the resulting PCR products were digested with EcoRI/ApaI and ApaI/XbaI, respectively, and then cloned into pQE31 digested with EcoRI/XbaI. For the construct pQE-C63KK/A, a slightly different strategy was used. The same PCR products were cut with EcoRI/ApaI and ApaI/SalI (the SalI site is present in the original gene sequence) and then ligated into plasmid pQE-C63K cut with EcoRI/SalI. All plasmids were transformed into Escherichia coli XL-1 cells. All mutants were verified by restriction analyses and sequencing.
Expression, purification of His-tagged recombinant TGBp1, SDS-PAGE and Western blot analyses of proteins.
E. coli strain M15 transformed with the recombinant vectors was grown at 37 °C in liquid culture until an OD600 of 0.8–0.9 was reached. Expression of the proteins was induced with 1 mM IPTG followed growth for 2–3 h at 37 °C. The purification of recombinant proteins from cultures was performed using a general procedure described by the manufacturer (Qiagen) for denaturing Ni-NTA chromatography. Proteins were fractionated by SDS-PAGE and stained with Coomassie blue. In several experiments, proteins were transferred onto a nitrocellulose membrane after electrophoresis and probed in a standard Western-blot assay using polyclonal rabbit antibodies raised against the recombinant 25K and 63K proteins.
ATP hydrolysis assay.
Reaction mixtures contained 10 mM Tris/HCl, pH 8.0, 10 % glycerol, 1 mM DTT, 5 mM MgCl2, 1 µCi [
-32P]ATP (6000 Ci mmol–1) and between 50 and 300 ng dialysed protein in a final volume of 10 µl. Probes were incubated for 1 h at 37 °C and the reaction was stopped by the addition of EDTA to a final concentration of 20 mM. To estimate ATPase activity, the unreacted ATP was precipitated by the addition of 200 µl 7.5 % activated charcoal in 50 mM HCl/5 mM H3PO4; the mixtures were vortexed and allowed to stand for 5 min and the charcoal was then eliminated by centrifuging for 10 min. Half of the supernatant was then analysed by Cherenkov counting for free [32P]phosphate released by hydrolysis of the labelled ATP. The sample incubated without protein was used as a negative control. The total sample activity without incubation and precipitation (minus a negative control sample) was taken as 100 %. ATPase activity of the proteins was expressed as relative activity (percentage hydrolysis of 32P-labelled substrate).
Kinetic parameters for ATP hydrolysis were measured in the linear phase of the reaction as described previously (Rakitina et al., 2005). Briefly, the standard [-32P]ATP-containing reaction mixture was supplemented with 100 or 200 ng protein and different concentrations of unlabelled ATP varying from 5 to 70 µM and 1 µCi (8 nM) [-32P]ATP. After 30 min incubation, ATP hydrolysis was analysed using the activated charcoal method as described above. Lineweaver–Burke double-reciprocal plots were used to calculate the Km and Vmax of ATP hydrolysis.
RNA-binding assay (gel-shift assay).
RNA (0.5 µg) from Tobacco mosaic virus (TMV) was incubated with various concentrations of protein for 15–30 min at room temperature in 15 µl gel-shift buffer (10 mM Tris/HCl, pH 8.0, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT and 50 mM NaCl). Samples were analysed in ethidium bromide-containing 1 % agarose gels in 1x Tris/acetate buffer. Application of Hill transformations to the RNA-binding data allows calculations of the Hill coefficient (an indicator of the cooperativity of RNA-binding; Marcos et al., 1999). The dissociation constant (Kd) for the protein–RNA interaction, defined as the concentration of the protein at half saturation (mid-point), was also determined on the basis of these data.
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RESULTS |
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). To analyse further the enzymic activity of the PVX TGBp1, also termed ‘25K protein’, we constructed a deletion mutant lacking the C-terminal region, including motifs III–VI. The resulting truncated protein, referred to as the 25KI–II mutant, comprised the 96 N-terminal amino acid residues of the 25K protein including the conserved motifs I, Ia and II of the NTPase/helicase domain (Fig. 1a). The mutant and the full-length wild-type 25K proteins were expressed in E. coli as fusions with an N-terminal 6x His tag and purified by Ni-NTA chromatography. Under standard conditions of the ATPase assay, the 25KI–II and wild-type 25K proteins demonstrated similar activity (Fig. 2a). On the other hand, the previously described mutants 
CIII-25K and N-25K (Fig. 1a) (Morozov et al., 1999) were enzymically inactive (Fig. 2a), as expected. The 25KI–II mutant has been constructed by engineering a translational terminator in place of the codon for amino acid residue 97 of the wild-type 25K protein, while in the CIII-25K mutant, a frame-shift mutation was introduced after residue 84 (Fig. 1a; Morozov et al., 1999). Thus, the frame-shift mutation in the CIII-25K mutant is located exactly at the NTPase/helicase motif II and the 25K-specific sequence in the ATPase-deficient mutant CIII-25K is 12 amino acid residues shorter than the enzymically active 25KI–II mutant. (Fig. 1a). Therefore, since further C-terminal truncations of the 25KI–II mutant destroy sequences essential for ATPase activity, we suppose that this mutant contains a minimal set of structural elements required for ATP hydrolysis by the 25K protein. These data point to the fact that the N-terminal region of the TGBp1 is sufficient for proper folding and functioning of the ATPase active centre.
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). Based on the C63K protein, a C-terminally truncated C63KI–II mutant was constructed (Fig. 1b
), which comprised the protein region including the NTPase/helicase motifs I, Ia and II (113 amino acid residues extending from residue 291 to 403 of the full-length 63K protein). Therefore, this mutant was structurally similar to the 25KI–II mutant. The C63KI–II mutant exhibited ATPase activity similar to that of the C63K protein, although the N-63K mutant (Fig. 1b) (Kalinina et al., 2002) was enzymically inactive (Fig. 2b). Thus, a portion of the TGBp1 proteins of both virus types including three NTPase/helicase motifs, I, Ia and II, and a short sequence upstream of motif I retained ATPase activity. NTPases/helicases of SF1 are known to have high intrinsic ATPase activity and, unlike superfamily 2 helicases, they can be stimulated by the presence of RNA by no more than 2-fold. In our case, no essential differences were evident in the ATPase activity of the mutant proteins in the presence or absence of RNA, and ATPase activity of the full-length and truncated mutants was stimulated by 1.4- to 1.7-fold (data not shown).
To characterize the ATPase activities of the 25KI–II and C63KI–II mutants in more detail, we measured the kinetic parameters of ATP hydrolysis. The Km for ATP hydrolysis and an apparent Vmax value were determined as described by Rakitina et al. (2005). For both the 25KI–II and C63KI–II mutants, C-terminal truncation of the NTPase/helicase domain resulted in slightly increased Km values, compared with the 25K and C63K proteins (Table 1). The Km of ATP hydrolysis for the 25K protein was 11±1.5 µM. Deletion of the C-terminal half of the 25K protein affected the Km only slightly (13±2.0 versus 11±1.5 µM), and a similar pattern was observed with the helicase domain of the 63K protein. The Km for the C63K protein was 14±1.2 µM, compared with 16±1.5 µM for the truncated C63KI–II mutant. Thus, in both potex-like and hordei-like TGBp1s, the affinity for ATP at the ATPase active site was similar for the full-length NTPase/helicase domain and for its isolated N-terminal region retaining motifs I, Ia and II. Apparent Vmax values were 3.8±0.2 and 4.0±0.25 nmol min–1 mg–1 for the full-length 25K and C63K proteins and 16.0±0.5 and 14.0±0.4 nmol min–1 mg–1 for the 25KI–II and C63KI–II mutants, respectively (Table 1). Since an apparent Vmax is calculated per mg protein, and the molecular masses of the 25KI–II and the C63KI–II mutants are about half the molecular masses of the 25K and C63K proteins, the molar activity, which reflects the velocity of ATP hydrolysis, is approximately 2-fold higher for the truncated mutants compared with the full-length helicase domains.
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). In particular, equal aliquots of non-specific unlabelled RNA (virion RNA of TMV) were incubated with increasing amounts of protein and then subjected to electrophoresis in ethidium bromide-containing agarose gels and visualized under UV light. In control experiments, protein–RNA complexes formed by the 25K protein were unable to enter the gel, and all of the RNA was incorporated in such complexes at a protein : RNA molar ratio close to 200 : 1 (Fig. 3a), in agreement with our earlier data (Kalinina et al., 2001). Similar patterns of protein–RNA interactions were found for the 25KI–II mutant, except that fully retarded complexes were formed at a higher protein : RNA molar ratio of approximately 400 : 1 (Fig. 3a). Comparison of RNA binding of the C63KI–II and C63K proteins revealed similar shifts in protein : RNA ratios. All RNA appeared to be bound to the protein at molar ratios of about 140 : 1 for the C63K protein and 350 : 1 for the C63KI–II mutant (Fig. 3b). The N-terminally truncated N-25K and N-63K mutants were incapable of binding RNA (Kalinina et al., 2001; Fig. 3). Thus, the N-terminal part of the helicase domain of both the 25K and 63K proteins seems to be primarily responsible for interactions of this domain with RNA.
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). Slightly increased Hill coefficients indicate that the truncated mutants retained the ability to bind RNA with cooperativity similar to the wild-type helicase domains. No significant differences in protein–RNA interactions could be found under high salt concentrations (200 and 400 mM NaCl; data not shown). Gel-retardation experiments were also carried out with a double-stranded RNA of about 300 bp; there were no significant differences in the efficiency of binding between single-stranded and double-stranded RNA (data not shown).
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; Liou et al., 2000; Lin et al., 2004). One of these residues, which corresponds to Arg-15 in the PVX 25K protein and Lys-315 in the PSLV 63K protein, is conserved among both potex-like and hordei-like TGBp1s (Morozov & Solovyev, 2003). To study the functional role of these residues, Arg-15 in the 25K protein and Lys-315 in the C63K protein were replaced by Ala, as well as in the respective C-terminally truncated 25KI–II and C63KI–II mutants. The ATPase activity and RNA-binding properties of the resulting mutants 25KR/A, 25KR/AI–II, C63KK/A and C63KK/AI–II (Fig. 1) were analysed.
It was found that the ATPase activity of the 25KR/A and C63KK/A point mutants, comprising the full-length NTPase/helicase domains, decreased considerably compared with that of the 25K and C63K proteins (Fig. 5a, b). The Km values for the 25KR/A and C63KK/A mutants were 26±2.0 and 30±1.1 mM, respectively. The apparent Vmax was 0.6±0.2 and 0.4±0.1 nmol min–1 mg–1 compared with 3.8±0.2 and 4.0±0.25 nmol min–1 mg–1 for the full-length 25K and C63K proteins (Table 1). Thus, the velocity of ATP hydrolysis is about 10-fold lower than that of the wild-type proteins.
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; Table 1). These data suggest that the introduced substitutions are unlikely to have a direct effect on the intrinsic enzymic activity of TGBp1 ATPase per se.
Quantitative evaluation of gel-shift RNA-binding assay data showed no significant difference in Kd between these mutants and the wild-type proteins (data not shown). Interestingly, the cooperativity of RNA binding (determined as Hill coefficients) for the full-length (25KR/A and C63KK/A) and truncated point mutants (25KR/AI–II and C63KK/AI–II) decreased notably compared with the proteins without point mutations (Fig. 4).
Role of the N-terminal part of the TGBp1 NTPase/helicase domain in protein self-interactions
The recombinant 25K protein, as well as the C63K protein, typically migrated in SDS-PAGE as a single band (Kalinina et al., 1996, 2001; Fig. 6a). Interestingly, the 25KI–II and 25KR/AI–II mutants reproducibly gave two bands. The lower band had the size expected for the recombinant protein, whereas the size of the upper band corresponded to the protein dimer (Fig. 6a). In contrast, the deletion mutants CIII-25K and N-25K both migrated as single bands (Fig. 6a). These observations indicate that the C-terminally truncated mutants 25KI–II and 25KR/AI–II formed stable dimers in the presence of SDS. Importantly, the intrinsic ability of the isolated TGBp1 N-terminal region for stable self-interaction was confirmed by observation of protein dimers formed by the C63KI–II and C63KK/AI–II mutants (Fig. 6a). Although this is unusual, such enormously stable self-dimers of different proteins have been previously reported by others (Bentley et al., 2002; Gentile et al., 2002; Taliansky et al., 2003).
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). Therefore, the N-terminal region of the TGBp1 NTPase/helicase domain could account for homologous protein interactions. |
DISCUSSION |
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). The consensus Walker A (G/AXXXXGKT/S, where X is any amino acid) and Walker B (ZZZZD or ZZZZDE, where Z is a hydrophobic amino acid) motifs are included in a common structural 
-sheet core for numerous proteins with ATPase activity (Koonin, 1993; Leipe et al., 2002; Vetter & Wittinghofer, 1999). Structurally, P-loop NTPases can be subdivided into several lineages. TGBp1 proteins belong to the RecA/F1-ATPase superclass and, more specifically, fall into helicases of SF1 (Koonin, 1993; Koonin & Dolja, 1993; Leipe et al., 2002). Site-directed mutagenesis and X-ray crystallographic studies revealed that, among the RecA/F1 superclass, F1-ATPases possess the minimal set of motifs required for NTP hydrolysis. These motifs represent Walker A and B sites as well as catalytically important signatures (Lobau et al., 1997). It is known that the lysine amino group interacts with MgATP/MgADP phosphates, that the serine or threonine hydroxyl group coordinates a Mg2+ ion (Walker A site), that an aspartic acid in position 1 coordinates a Mg2+ ion and that a glutamic acid in position 2 is a putative catalytic residue (Walker B site) (Caruthers & McKay, 2002). In this paper, we used deletion mutants of the PVX 25K and PSLV 63K proteins (Fig. 1) to demonstrate that the N-terminal region of the TGBp1 NTPase/helicase domain, which contained only three conserved NTPase motifs [I (Walker A), Ia and II (Walker B)], was sufficient for the ATPase function (Fig. 2a, b). Despite evident sequence similarities between the SF1 RNA helicases (including TGBp1) and the SF1 DNA helicases (Koonin, 1993; Koonin & Dolja, 1993), these proteins appear to display different structural requirements for catalysis of ATP hydrolysis. The NTPase activity of DNA helicases was shown to depend not only on motifs I, Ia and II, as was found for TGBp1s, but also on other conserved motifs of the NTPase/helicase domain (Caruthers & McKay, 2002). However, mutational analysis of herpes simplex virus UL5 helicase, also belonging to SF1, showed that changes in motifs III, IV, V and VI did not eliminate ATPase activity of the protein or affect DNA binding. Nevertheless, these mutants completely lacked helicase activity (Graves-Woodward et al., 1997). In 1996, Koonin and Rudd, using computer methods for protein sequence analysis, suggested that the N- and C-terminal domains of SF1 helicases have distinct activities: only the N-terminal domain possesses ATPase activity (Koonin & Rudd, 1996). Later, we confirmed this suggestion experimentally by showing that the ATPase activity of the PVX TGBp1 is not inhibited, but is rather stimulated, by deletion of C-terminal motifs V and VI (Morozov et al., 1999). In the present work, we have defined the enzymically active region for the helicase domains of two types of TGBp1 proteins and have shown that the ‘minimal ATPase subdomain’ includes only three of seven conserved motifs.
In addition to ATPase activity, the TGBp1 NTPase/helicase subdomain comprising motifs I, Ia and II retained nucleic acid-binding activity (Fig. 3), which is in agreement with previous data (Kadare et al., 1996; Morozov et al., 1999). BaMV TGBp1 has been shown to have three basic amino acid residues in the region upstream of motif I that are important for RNA binding (Wung et al., 1999; Liou et al., 2000). Our data revealed no significant influence of the conserved basic amino acid residue upstream of motif I on the RNA-binding activity of helicase domains of TGBp1s. It is possible that motif Ia and the following minimally conserved TXGX motif take part in nucleic acid–protein interactions, as has been shown for DNA helicases (Caruthers & McKay, 2002). According to our data, the RNA-binding activity of TGBp1 showed pronounced protein–protein cooperativity (Fig. 4), suggesting involvement of TGBp1 in homologous interactions. Indeed, the truncated mutants were able to form significant amounts of stable dimers even under SDS-PAGE conditions (Fig. 6a). Immunoblot analysis revealed that the full-length helicase domain of the PVX and PSLV TGBp1 proteins is capable of forming dimers and oligomers under mild sample treatment conditions (Fig. 6b). In addition, we found that the PVX TGBp1 is capable of self-interactions in a yeast two-hybrid system (unpublished data). Fractionation of the recombinant 25K and C63K proteins and their mutants in sucrose-density gradients revealed that the proteins could not be detected as monomers and appeared in the form of dimers and oligomers (to be published elsewhere). These observations are in agreement with earlier data on dimer formation in vitro by another potex-like TGBp1 (Wung et al., 1999; Liou et al., 2000) and homologous interactions of hordei-like TGBp1 in the yeast two-hybrid system (Cowan et al., 2002). TGBp1 homologous interactions are consistent with the general notion that many helicases form homodimers or oligomers (Gorbalenya & Koonin, 1993). For example, the activity of TMV replication helicase, which is very closely related to TGBp1 in its amino acid sequence, is dependent on homohexamer formation (Goregaoker & Culver, 2003).
Positively charged residues upstream of motif I in the potex-like TGBp1 have been shown previously to be essential for ATPase activity (Wung et al., 1999; Liou et al., 2000). In this paper, we evaluated the functional significance of one of the residues that is conserved in both potex-like and hordei-like TGBp1 proteins. Point mutations of this residue caused a substantial decrease in the ATPase activity of the full-length NTPase/helicase domain. However, these mutations failed to affect the ATPase activity of C-terminally truncated mutants comprising motifs I, Ia and II (Fig. 5). Point mutations reduced the protein cooperativity slightly in protein–RNA interactions (Fig. 4), but did not affect the ability of the mutants to form dimers in SDS-PAGE (Fig. 6a). Thus, we hypothesize that the conserved basic amino acid residue upstream of motif I plays an important role in maintenance of a reaction-competent conformation of the ATPase active centre in the full-length helicase domain. This confirms recent findings that point mutations outside the conserved motifs of the NTPase/helicase domain, presumably affecting protein conformation, have drastic effects on PVX TGBp1 activities in vitro (Bayne et al., 2005).
To conclude, we have observed that complete deletion of motifs III–VI in TGBp1 does not inhibit ATPase activity or RNA binding. We also show an important role for the conserved lysine/arginine residue upstream of the Walker A motif in the ATPase activity of potexviral and hordeiviral TGBp1 proteins. Based on these data, the ‘minimal ATPase subdomain’ of TGBp1 proteins has been determined. The engineered PVX and PSLV polypeptide derivatives of TGBp1 represent one of the smallest known proteins to display ATP hydrolysis, RNA binding and the capacity for homologous interactions.
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ACKNOWLEDGEMENTS |
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Received 22 February 2006;
accepted 23 May 2006.
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) and the truncated 25KI–II (
), 
) mutants. (b) ATPase activity of the PSLV C63K protein (
