Structural Properties of Carnation Mottle Virus p7 Movement Protein and Its RNA-binding Domain*

Marçal VilarDagger §, Vicent EsteveDagger , Vicente Pallás, Jose F. Marcos||, and Enrique Pérez-PayáDagger **

From the Dagger  Departament de Bioquímica i Biologia Molecular, Universitat de València, E-46100 Burjassot, València, Spain, the  Instituto de Biología Molecular y Celular de Plantas UPV-Consejo Superior de Investigaciones Científicas, Universidad Politécnica de València, Avda. de los Naranjos s/n, E-46022 València, Spain, and the || Departamento de Ciencia de los Alimentos, Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas, Apartado de Correos 73, Burjassot, E-46100 València, Spain

Received for publication, January 25, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plant viral movement proteins (MPs) participate actively in the intra- and intercellular movement of RNA plant viruses to such an extent that MP dysfunction impairs viral infection. However, the molecular mechanism(s) of their interaction with cognate nucleic acids are not well understood, partly due to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding properties of this class of proteins, as exemplified by the 61-amino acid residue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectroscopy showed that CarMV p7 is an alpha /beta RNA-binding soluble protein. Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p717-35), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent alpha -helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide). Thus, the RNA recognition appears to occur via an "adaptive binding" mechanism. Interestingly, the amino acid sequence and structural properties of the RNA-binding domain of p7 seem to be conserved among carmoviruses and some other RNA-binding proteins and peptides. The low conserved N terminus of p7 (peptide p71-16) is unstructured in solution. In contrast, the highly conserved C terminus motif (peptide p740-61) adopts a beta -sheet conformation in aqueous solution. Alanine scanning mutagenesis of the RNA-binding motif showed how selected positive charged amino acids are more relevant than others in the RNA binding process and how hydrophobic amino acid side chains would participate in the stabilization of the protein-RNA complex.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Infection of plants by viruses requires viral genome cell-to-cell movement through plasmodesmata (the plant intercellular symplastic connections), which is mediated by virus-encoded so-called movement proteins (MPs)1 (1, 2). MPs participate actively in the intra- and intercellular movement of plant viruses, and mutant virus analyses, reverse genetics, and plant transformation have demonstrated that MP dysfunction impairs viral infection (3, 4). MP properties are best described in the tobacco mosaic tobamovirus (TMV) model system. The 30-kDa TMV MP binds single-stranded (viral) RNA in vitro with no sequence specificity and high cooperativity (5), co-localizes with the cytoskeleton and cell wall (6-8), is required for the association of viral RNA with endoplasmic reticulum (9), increases the size exclusion limit of plasmodesmata (10), and mediates an active transport of the viral genome to the adjacent cell (4). Sequence deletion and mutagenesis studies have located some of these functions in separate motifs/domains of the protein (11-13). These properties define the tobamo-like model of viral cell transport. TMV MP names the "30K" superfamily of virus MPs that comprise proteins from 18-20 groups of viruses (14, 15) including viruses that move as tobamoviruses but also tubule-forming viruses (1). RNA (or DNA) binding properties are known for an increasing number of MPs that belong to taxonomically very diverse viruses, both within and outside those included in the 30K superfamily.

MPs appear, therefore, as a separate and functionally important group of proteins capable of binding RNA and which are critical for plant infestation. Strikingly, recent data suggest that viral MPs mimic plant proteins involved in an RNA-based long distance signaling system that would operate through plant vascular tissue (16). Antigenic cross-reaction (17) and sequence homology searches (15) have identified MP-like candidates among plant phloem sap proteins.

Progress has been made in the structural characterization of sequence-specific RNA-binding proteins and peptides and of their interaction with cognate RNAs (18, 19). Unfortunately, knowledge lags behind in the structural analyses of sequence-nonspecific RNA-binding proteins. Recently, TMV MP was shown to fold as an alpha -helical membrane protein in the presence of urea and SDS (20), and a topological model for its insertion into the cell membrane was proposed. Despite their hallmark functions, detailed experimental data on the structure of MPs is lacking, and the mechanisms of RNA recognition and structural parameters involved in the interaction with viral nucleic acids are far from being understood. An open question remains, for instance, as to the mechanism(s) that allow MPs (and plant MP-like proteins) to recognize and transport specific RNAs despite their demonstrated nonspecific in vitro RNA binding (21). Moreover, the study of proteins having membrane-spanning domains as MPs is hampered by the problems encountered in purification and/or solubilization in native conformation. An alternative approach would be to identify simplified polypeptide models of MP function, capable of insertion into biological membranes and/or RNA binding as water-soluble domains. Such models have not been found so far for viral MPs.

Alternatively, nature can be searched for examples in which MP functions map in separate proteins. There are viruses that encode multiple MPs, which, in addition, do not fall in the 30K superfamily. The triple gene block of potexviruses encodes three proteins (open reading frames 2-4) that mediate cell-to-cell transport, the first one with RNA-binding and NTPase/helicase activities and the other two with putative membrane-spanning domains and, in the case of the third, demonstrated cell wall localization (22, 23). Carmoviruses are among the smallest known plant viruses; their genome is a single-stranded RNA of ~4 kilobases encoding at least five proteins (24, 25). Carmoviruses also code for separate MPs, and the two corresponding genes have been considered as a "truncated triple block" (26). The type member of the group, carnation mottle virus (CarMV), encodes two very small overlapping polypeptides, p7 and p9, whose homologs in turnip crinkle virus have been shown to be involved in intercellular movement (27, 28). We have previously confirmed that CarMV p7 has RNA binding properties in vitro and demonstrated that a 19-amino acid peptide derived from p7 was likewise capable of binding to RNA (29). On the other hand, sequence analyses on CarMV p9 suggest the presence of two membrane-spanning domains. It was proposed that their small size made carmovirus MPs a suitable model for the structural characterization of MP and their interaction with viral RNA.

In the present study, we conducted a structural characterization of CarMV p7, and we used a retrostructural approach with the analysis of three separate individual peptides that cover most of the p7 sequence. We mapped the RNA-binding domain of the protein and showed its folding into a partial alpha -helix upon interaction with RNA. Finally, a series of amino acid residue substitutions were conducted on this domain in order to establish structure/RNA binding function relationships for this important class of proteins.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Materials-- Fmoc-protected amino acids for peptide synthesis were from SENN Chemicals (Gentilly, France). RNA in vitro transcription kit was from Promega, and the digoxigenin detection kit was from Roche Molecular Biochemicals. Deuterium oxide (2H2O) and trifluoroethanol (TFE)-d3 was from Cambridge Isotopes Laboratories (Cambridge, United Kingdom). All other chemicals were from standard suppliers.

Heterologous Production, Purification, and Refolding of p7-- p7 movement protein was produced and purified as in Ref. 29. Further purification was achieved by semipreparative RP-HPLC in a C-18 Lichrosphere analytical column (Waters, Barcelona, Spain). Fractions containing p7 protein were freeze-dried. A refolding protocol was carried out with the protein powder. First, the protein was solved at 1 mg/ml in buffer A (8 M guanidinium-HCl, 10% sucrose, 50 mM glycine, 1 mM EDTA, and 0.01 M NaOH, pH 5.5). The solution was dialyzed overnight against buffer B (8 M urea, 10% sucrose, 50 mM glycine, 1 mM EDTA, and 0.01 M NaOH, pH 5.5). Successive dialyses were carried out against decreasing concentrations of urea (4, 2, and 0 M) in the same buffer B. Finally, the protein was dialyzed against 10 mM phosphate buffer, pH 5.5. This p7 protein solution was used in CD experiments as described below.

Peptide Synthesis-- Peptides were manually synthesized by solid-phase peptide synthesis using Fmoc chemistry (30) as in Ref. 29, except for peptide p71-16, which was obtained from DiverDrugs (Barcelona, Spain). Analytical RP-HPLC and laser desorption time-of-flight mass spectrometry were used to determine the purity and identity of the peptides.

Circular Dichroism Spectroscopy-- All measurements were carried out on a Jasco J-810 CD spectropolarimeter, in conjunction with a Neslab RTE 110 water bath and temperature controller. CD spectra were the average of a series of 10 scans made at 0.2-nm intervals. CD spectra of the same buffer (or in the presence of TFE or SDS as described in the figure legends) without peptide were used as base line in all of the experiments. For p7 and peptides containing a tryptophan amino acid residue, the concentration was determined by UV spectrophotometry at 280 nm using epsilon Trp = 5570 M-1 cm-1. The concentration of all of the other peptides was obtained by quantitative amino acid analysis. The CD spectra of the p717-35-RNA complex were recorded at 5 °C with 20 µM of p717-35, and increasing concentrations of CarMV ssRNA transcribed in vitro from clone pCarM.D5 (Ref. 29; this RNA covers exactly the first 181 residues of the CarMV genome) were added (from 1 to 20 ng), until no change in the difference spectrum was observed. The difference spectrum was the RNA-peptide complex minus the free RNA spectrum. Spectra were recorded in the far UV region in 5 mM MOPS/NaOH, pH 7.0, buffer. In peptide tritation experiments, increasing concentrations of p717-35 were added (from 0 to 200 µM) to a 1 mM RNA solution and left on ice for 30 min before the CD spectrum was recorded in the near UV region (250-350 nm).

Electrophoretic Mobility Shift Assay (EMSA) Experiments-- Different amounts of either p7 or the synthetic peptides were incubated with 1 ng of pCarM.D5 ssRNA probe (29), in a 10-µl final volume of binding buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 5 units of HRPI RNase inhibitor (Promega) and 10% glycerol) for 30 min on ice. After incubation, 2 µl of loading buffer was added, and the samples were electrophoresed through a 1% agarose in TAE buffer (40 mM Tris-acetate, 2 mM EDTA) at 50 V for 1 h at 4 °C. RNAs were transferred to positively charged nylon membranes by capillary elution in 20× SSC and fixed to the membranes by UV irradiation on a UV gene linker for 2 min. Membranes were hybridized to a digoxigenin-labeled DNA probe overnight at 60 °C in PSE (7% SDS, 1 mM EDTA, 0.3 M sodium phosphate, pH 7.2). Detection of digoxigenin-labeled nucleic acids was conducted as described previously (29, 31). The amount of free RNA probe in each sample was quantified by densitometry of the films (Quantity One software, Bio-Rad).

NMR Spectroscopy-- NMR spectra were recorded on a Bruker Avance spectrometer operating at 500 MHz. Samples were prepared at 2.6 mM peptide concentration in two solvents: pure water and TFE/water (1:1, v/v). The spectra were acquired at 283 K. Water signal suppression was achieved using the WATERGATE sequence (32). Phase-sensitive double quantum filtered COSY (33), TOCSY (34), and ROESY (35) were used for sequence-specific assignments. Heteronuclear single-quantum coherence (36) and heteronuclear multiple quantum correlation (37) experiments were performed to assign alpha C chemical shifts. TOCSY and ROESY spectra were recorded using the MLEV-17 spin-lock sequence. Mixing times for TOCSY spectra were 15 and 80 ms. Mixing times for ROESY experiments were 250 and 300 ms. The number of scans varied between 32 and 64, the number of t1 increments varied between 512 and 1024, and the number of points in the t2 dimension was 2048. 1H resonances were assigned using standard procedures (38).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification and Secondary Structure Characterization of Three Different Structural Domains in CarMV p7-- In our previous study, the p7 movement protein from a Spanish isolate of CarMV was sequenced and produced in bacteria, and its in vitro RNA binding properties were demonstrated (29). In order to confirm that the previously described RNA-binding peptide (29) corresponds to the binding domain of p7, this peptide (p717-35) and two additional peptides (p71-16 and p740-61) derived from the primary sequence of p7 (Fig. 1A) were synthesized and assayed for RNA binding. Fig. 1B shows the EMSAs for the three peptides. As the concentration of p717-35 was increased, the amount of free viral RNA decreased concomitant with the appearance of a low electrophoretic mobility band that corresponds to an RNA-p717-35 complex. Our data demonstrated that the N-terminal and C-terminal amino acid sequences of the p7 protein did not bind to RNA as shown by the lack of RNA-peptide complex formation. Therefore, we have unambiguously located the p7 domain responsible for the RNA binding in the amino acid motif covered by peptide p717-35.


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Fig. 1.   RNA binding properties of synthetic peptides derived from CarMV p7. A, amino acid sequences of the CarMV p7 MP and their derivative peptides. In the p7 sequence, the distribution of negative (-) and positive (+) charge residues is labeled. Conserved residues among carmovirus homologous proteins are highlighted with an asterisk. The amino acid sequence of three synthetic peptides derived from p7 (p71-16, p717-35, and p740-61) is shown as well their location in the amino acid sequence of p7. B, EMSA for RNA binding of peptides p71-16, p717-35, and p740-61. Increasing concentration of peptides (up to 50 µM) were incubated with ssRNA probe (see "Experimental Procedures").

The secondary structures of CarMV p7 and the three synthetic peptides were analyzed by means of CD spectroscopy. Recombinant viral MPs typically form insoluble inclusion bodies that facilitate purification procedures (5, 39). However, protein recovered from solubilized inclusions must be refolded to native conformation before biophysical characterization. We have developed a procedure for refolding of His-tagged CarMV p7 purified from bacteria that provided soluble and functional protein capable of RNA binding (see "Experimental Procedures"). The far UV CD spectrum of purified and refolded CarMV p7 (Fig. 2A) changed as compared with nonrefolded protein (data not shown), and it was evaluated for secondary structure content showing 43% unordered structure, 5% turns, and 10 and 42% alpha -helical and beta -sheet structure, respectively.


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Fig. 2.   Far UV CD spectra of the p7 MP and peptides p71-16, p717-35, and p740-61. A, CD spectrum of refolded p7 MP acquired at a protein concentration of 20 µM in 10 mM phosphate buffer, pH 7.5, at 25 °C. B, C, and D, CD spectra of peptides p71-16, p717-35, and p740-61, respectively, in different experimental conditions. Solid line, in 5 mM MOPS/NaOH pH 7.0 buffer solution; dotted line, in the presence of 50% TFE; dashed line, in the presence of 1 mM SDS.

The spectra for the synthetic peptides derived from the p7 protein displayed remarkably distinct shapes. p71-16 and p717-35 showed an apparent unordered structure (Fig. 2, B and C), while p740-61 showed a CD spectrum typical for a beta -sheet structure (Fig. 2D). In order to assay the propensity of each peptide to be induced into a defined secondary structure, their CD spectra were also recorded in the presence of TFE, a solvent know to induce helicity in single-stranded potentially alpha -helical polypeptides (40) or submicellar concentrations of SDS, which is considered a template that would stabilize both alpha -helical or beta -sheet conformations depending on the intrinsic propensity of the polypeptide sequence to adopt a preferred secondary structure (41). In 50% TFE, p71-16 and p717-35 peptides were induced into an alpha -helix with different mean residue ellipticities (Fig. 2, B and C). In the presence of 1 mM SDS, however, p71-16 was unordered (Fig. 2B), while p717-35 was again induced into an alpha -helix (Fig. 2C). In contrast, the CD spectra obtained in buffer for p740-61 was not significantly modified by the presence of either 50% of TFE (Fig. 2D) or SDS (data not shown).

From these results, we conclude that the central region of p7, as represented by p717-35, is an inducible alpha -helix that is responsible of the RNA binding of p7 to ssRNA, while its C-terminal region (p740-61, which represents 31% of the amino acid sequence of p7) folds into a highly stable beta -sheet that would account for the overall beta -sheet content of p7.

Structural Changes Occurred upon p717-35 Peptide-Viral RNA Complex Formation-- CD spectroscopy has been previously used to report structural changes in peptides that bind to RNA (42, 43). The CD difference spectrum resulting from p717-35 peptide-viral RNA complex formation (RNA-peptide complex minus free RNA) showed significant changes in the far-UV region as compared with that of the free peptide (Fig. 3A). Upon binding to CarMV RNA, the peptide p717-35 showed a conformational change characterized by a decrease in the random contribution at 200 nm and an increase in ellipticity at 220 nm (Fig. 3A). These changes in the CD spectrum are consistent with an increase in helical content in the secondary structure of the peptide. In addition, the RNA was constrained due to the formation of the complex. The CD spectrum in the absence of peptide showed a positive band with a maximum intensity at 270 nm characteristic of ssRNA; however, as the concentration of p717-35 was increased, a decrease in the CD signal and a red shift was observed (Fig. 3B). This type of change has been attributed to an unstacking of RNA bases as a consequence of peptide binding (44). These results imply that not only did the peptide change its conformation upon binding to RNA but also the RNA adapted its conformation to allow the stabilization of the RNA-peptide complex.


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Fig. 3.   CD analysis of the interaction of p717-35 peptide with ssRNA. A, far UV CD spectra of p717-35 peptide (solid line) and of p717-35-RNA complex (dotted line, spectra obtained as described under "Experimental Procedures"). B, near UV CD spectra of a 1 mM concentration CarMV ssRNA upon binding to p717-35. From the top, the peptide concentrations added were 0, 10, 20, 100, and 200 µM.

NMR Structural Characterization of p717-35-- The CD secondary structure characterization of p7 and p717-35, together with the RNA binding experiments, allowed us to define the peptide p717-35 as an alpha -helical inducible protein motif. There are many examples in the literature of RNA-binding peptides and/or protein domains that fold in an alpha -helix upon binding (45) and even some within plant virus coat proteins (46), but this is the first example on a MP. Moreover, the secondary structure prediction using AGADIR (47) suggested that the segment with the highest tendency to adopt an alpha -helical structure in p7 is 24TRRSVAKDAIRK35 (data not shown). In order to confirm this putative alpha -helix, we carried out NMR analysis of the peptide p717-35. The NMR spectra for p717-35 was recorded in aqueous solution and in 50% TFE. Sequential assignments were obtained from the "fingerprint" region of the two-dimensional TOCSY, ROESY, and NOESY spectra. The adoption of a defined preferential secondary structure by a peptide induces significant and specific chemical shift changes in both 1H and 13C nuclei that can be used to quantify secondary structure populations. In particular, an alpha -helical conformation is characterized by upfield and downfield shifts of the alpha H and alpha C nuclei, respectively. Therefore, one can then measure, for each residue, the deviation of the experimental chemical shifts from those attributed to random coil conformations (38). The polypeptide region that comprises the seven C-terminal amino acids in p717-35, from Ala-29 to Lys-35, appeared to populate a nascent alpha -helical conformation in water solution that is further stabilized in the presence of 50% TFE as defined by the analysis of the conformational chemical shifts of 1Halpha and 13Calpha (Fig. 4; a schematic of the helix is shown in Fig. 5D). Although cross-peak superposition between sequential and medium range NOEs precluded a more detailed identification, a set of medium range NOEs typical of alpha -helix were found in water solution (dalpha N i, i + 3 between Ser-27 and Lys-30 and between Asp-31 and Arg-34; dalpha N i, i + 4 between Asp-31 and Lys-35, data not shown) corresponding with strings of dNN NOEs. Furthermore, additional dalpha N NOEs were identified in 50% TFE solution (i, i + 3 between Lys-30 and Ile-33 and between Ala-32 and Lys-35; i, i + 4 between Ala-29 and Lys-35 and between Lys-30 and Arg-34, data not shown).


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Fig. 4.   NMR analysis of peptide p717-35 in water and in the presence of 50% TFE. Observed conformational chemical shift increments for 1H (Delta delta 1 Halpha ) and for 13C (Delta delta 13Calpha ) relative to the chemical shifts of random peptides.


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Fig. 5.   A, sequence alignment of CarMV-dix p7 RNA-binding domain (this work; accession number AJ304989) and the homologous region of the other eight carmoviruses sequenced so far. CCFV, cardamine chlorotic fleck virus (accession number L16015); CPMoV, cowpea mottle virus (U20976); MNSV, melon necrotic spot virus (M29671); SgCV, saguaro cactus virus (U72332); TCV, turnip crinkle virus (M22445); GaMV, galinsoga mosaic virus (Y13463); HCRSV, hibiscus chlorotic ringspot virus (X86448); JINRV, Japanese iris necrotic ring virus (D86123). Conserved residues are shaded and shown as consensus sequence below. B, amino acid sequence alignment of CarMV p7 MP and Cucurbita maxima CmPP16-1 protein (18). The region of p7 MP that is folded into an alpha -helix and the putative homologous region of CmPP1-16 are boxed. C, sequence alignment of peptide p717-35 and a peptide derived from the N-antitermination protein of phage P22 (73). The location of the alpha -helix implicated in RNA binding in p717-35 (this paper) and in P22 peptide are shown as boxes. Residues important for RNA binding in both peptides are underlined. In B and C, identical residues are marked with an asterisk, conservative changes with a dot, and basic amino acids with boldface letters. D, side view (left) and top view (right) cartoon showing the amphipathic character of the alpha -helix identified at the C terminus of p717-35.

RNA Binding Analysis by Alanine-substituted Peptides-- To determine amino acid residues within the predicted alpha -helical segment of p717-35 that might be implicated (see Fig. 5D) in the RNA binding properties of the peptide, a series of p717-35 derivatives was synthesized containing a single alanine substituted at each position from Thr-24 to Lys-35, and their RNA binding potentials were evaluated by the apparent dissociation constant (kD) obtained after quantitation of EMSAs (Table I). Most of the peptides studied containing alanine substitutions at any of the five basic residues showed a lowered affinity for RNA (as measured by their higher kD; Table I). Mutations at Arg-34 and Lys-35 were found to have a major effect on RNA binding activity, while those at Arg-24, Arg-25, and Lys-30 had an intermediate contribution. These results suggest that these two basic amino acids are important for RNA binding of peptide p717-35, and probably for the whole p7 protein. In order to check whether the results obtained in EMSA experiments were only attributable to the specific amino acid side chain replacement and not to a structural change, we evaluated the ability of the peptides to be induced into an alpha -helical conformation. All five basic residue substitution analogs retained propensities to fold into an alpha -helix that did not differ significantly from each other or differ from that of the wild type p717-35 peptide (Table I). Thus, Arg-34 and Lys-35 mutations may remove direct RNA contacts rather than affecting binding through alterations of the structural propensity of the peptide. Arginine residues can form bidentate interactions with phosphates, sugars, and/or bases of the target RNA, whereas lysine residues may serve as hydrogen bond donors. It is worthy note that an arginine and/or a lysine residue is present at the end of the putative alpha -helix in all p7 homologous MPs of all carmoviruses (see Fig. 5A), reinforcing the critical role of these two amino acids in the RNA binding properties observed for CarMV p7 MP.

                              
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Table I
Amino acid sequence, RNA binding properties, and alpha -helical content of the alanine-scanning substitution analogs of p717-35

Replacement of Asp-31 by alanine induced an increase in the binding affinity of the peptide, most likely correlated with a lower repulsion to the negatively charged nucleic acid. Thus, this Asp residue decreases the RNA binding potential of the native sequence. This acidic amino acid residue, which is maintained in other carmovirus MPs in this position of the motif, could play a role in the fine modulation of RNA binding and also in the stabilization of an alpha -helical conformation in this polypeptide segment, as concluded from our NMR data (medium range NOEs, dalpha N i, i + 3 between Asp-31 and Arg-34; dalpha N i, i + 4 between Asp-31 and Lys-35). A recent study on the molecular variability of CarMV isolates from different geographical origins2 revealed that Asp-31 was conserved in all of the 22 isolates characterized.

RNA binding affinity by additional substitution analog peptides was examined to evaluate the importance of noncharged side chain residues. Thus, replacement of Thr-24, Ser-27, and Val-28 by alanine had an intermediate effect in RNA binding affinity (Table I). Nevertheless, when Ile-33 was replaced by alanine, the apparent dissociation constant for RNA binding markedly increased, although the ability of this peptide to fold into an alpha -helix in the presence of TFE was, again, similar to that of the wild type p717-35 peptide (Table I). A bulky aliphatic residue is present in a similar position to that of Ile-33 in all carmovirus MPs except for cardamine chlorotic fleck virus and turnip crinkle virus (Fig. 5A), strongly suggesting an important role in their RNA binding capabilities. The importance of hydrophobic contacts between RNA-binding domains and their cognate RNA has been previously reported (48). In particular, Chen and Frankel (49) have found that an isoleucine residue in a 17-amino acid peptide derived from the bovine immunodeficiency virus Tat protein is required for specific peptide binding to RNA.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The binding mechanism and subsequent cell-to-cell transport of plant viral RNA mediated by MPs is unsolved. MPs have been functionally characterized (50-52), but knowledge of the structural elements that determine their properties lags behind. Understanding the structural properties of this class of proteins will further contribute knowledge of both the mechanism of plant virus movement and the process of protein-RNA recognition. In the absence of three-dimensional data, the results presented here constitute a new approach in the understanding of the global conformation of a soluble plant virus MP.

The CD analysis of p7 spectra allowed us to estimate that recombinant p7 is structured after isolation from bacteria inclusion bodies, purification, and refolding. Due to the denaturing conditions used in the purification protocol, a refolding strategy was essential to obtain a highly soluble protein solution with no aggregation during storage. We showed that CarMV p7 MP is an alpha /beta -RNA-binding protein with an estimation of 42% beta -sheets, 10% alpha -helices, 5% turns, and 43% random coil. This folding in mixed alpha  and beta  structures is found in other RNA-binding proteins and also is predicted for MPs from the 30K superfamily of viral movement proteins, where a common core domain consists of two alpha -helices separated by beta -sheet structures (15). Although CarMV p7 MP does not belong to that family of MPs, it is interesting to note its structural similarity. Moreover, the high amount of beta -sheet structure in p7 is similar to that concluded from the CD analysis of single-stranded DNA (ssDNA)-binding proteins from phages and some prokaryotes (53, 54). ssDNA-binding proteins share some similarities with movement proteins in terms of binding to single-stranded nucleic acids in a highly cooperative process (55). The nucleic acid binding process of these proteins and MPs could be similar because it has been demonstrated that a ssDNA-binding protein competes with a MP for ssDNA binding in vitro and in vivo (56). Recently, the CD spectrum of TMV MP has been reported, showing a high percentage of alpha -helix (more than 70%) (20). In that study, the spectra were recorded in the presence of 0.1% SDS and 2 M urea, conditions required due to the high tendency to aggregation of TMV MP and that are markedly different from those used in our p7 CD measurements.

To help establish the location of the RNA-binding domain on the p7, we have used synthetic peptides that turned out to be also useful to unravel the conformational capabilities of isolated domains of CarMV p7 (Figs. 1 and 2). In previous work, we identified that a peptide derived from p7 was able to bind to RNA as the whole protein (29). In the present work, we have demonstrated that only this peptide has RNA-binding activity and that the flanking domains defined by peptides p71-16 and p740-61 did not bind to RNA (Fig. 1). The retrostructural approach used in the present study allows us to define three different putative domains in p7. The N-terminal domain, which covers amino acids 1-16, has a very low content of secondary structure, and it can be induced to fold into an alpha -helix only in the presence of strong secondary structure inducers like TFE and not in the presence of submicellar concentrations of SDS. This domain has the highest sequence variability when compared with other carmovirus MPs (29).2 The C-terminal domain is extremely conserved in terms of amino acid sequence (Fig. 1A). This domain showed a CD spectrum indicative of a defined beta -sheet secondary structure in aqueous solution that probably accounts for the overall beta -sheet content in the whole p7 protein. Due to its high sequence conservation, this domain should have a relevant functional role. It has been reported that when ssDNA-binding proteins, like gene 5 protein from Pf1, bind to ssDNA, a highly structured beta -sheet protein domain mediates the protein-protein interaction (57). It is tempting to speculate that this conserved beta -sheet domain could be responsible for the protein-protein interactions that confer cooperativity to the binding process of p7 to RNA (29).

The RNA-binding domain of p7 is unambiguously located within amino acids 17-35 (Fig. 1). The peptide that defines this domain in the present study (p717-35) has been structurally characterized as alpha -helical inducible (Fig. 2C). Like other RNA-binding peptides, it undergoes a conformational change upon RNA binding (Fig. 3) concomitantly with a conformational change in the secondary structure of the RNA. These features suggest that both the peptide and the RNA structures have to adapt to each other to stabilize the complex upon binding as has been described for other peptide-RNA complexes (43). In particular, conformational changes similar to that described in the present study have been reported for peptides derived from the HIV regulatory proteins Tat or Rev upon binding to their cognate HIV RNAs (58) and from the 25 N-terminal amino acids of cowpea chlorotic mottle virus coat protein after binding to its RNA (46). The structural flexibility of protein and RNA domains would favor the complete interaction of these macromolecules by a dual fitting mechanism. Such an induced fit may be needed to allow a set of specific contacts to form cooperatively with different amino acids of the peptide (conformational specificity) that would further induce a more pronounced secondary structure peptide stabilization. However, conformational specificity requires not only a stable folded state but probably also a partially defined prefolded stated in the unbound peptide (or protein). In fact, when analyzed by NMR, the p717-35 peptide shows a nascent alpha -helix conformation that covers amino acids 29AKDAIRK35. Nascent helices can form in short peptides, provided that amino acids either with high helical propensities or with stabilizing interactions between side chains are present (59). Also, it has been defined as a stabilizing factor the electrostatic interactions with the ends of the helix macrodipole by amino acids at the N or C terminus that cap the helix with additional hydrogen bonds (60). All of these factors seem to be present in the C-terminal end of p717-35. Alanine and arginine have high helical preference (61); alanine can also serve as N-terminal end capping, and positively charged amino acids have a favorable interaction with the helix C-terminal macrodipole. Furthermore, Asp-31, a negatively charged amino acid that is conserved among other carmovirus MPs, is placed in a positively charged amino acid context that could minimize the peptide intramolecular charge repulsion. This aspartic residue could stabilize the nascent alpha -helical conformation and could contribute to a fine modulation of the RNA binding activity of these MPs. In fact, our NMR experiments demonstrated that this amino acid might contribute to helix stabilization by salt bridge interaction with amino acid residues at i + 3 (Arg-34) and i + 4 (Lys-35) positions. In addition, a peptide analog where Asp-31 was substituted to alanine (p717-35D31A), showed an increased RNA binding activity. Consistent with this idea, it is remarkable the recent observation that strain B of turnip crinkle virus has a greater ability to spread in planta than the turnip crinkle virus-M strain, and this ability is due to the mutation of Lys-25 to Glu in turnip crinkle virus-B MP p8, which inversely correlates with p8 RNA binding affinity (62).

Amino acid residues implicated in the process of RNA binding were identified by alanine-scanning mutagenesis between Thr-24 and Lys-35. In general, positive charged amino acid mutations to alanine, p717-35-R25A, p717-35-R26A, p717-35-R34A, and p717-35-K35A, are important for RNA binding. Because of the non-sequence-specific RNA binding, this is consistent with an electrostatic attraction to the target RNA by these peptides. However, is noteworthy that not all basic residues contributed equally; thus, p717-35-R34A and p717-35-K35A mutants showed the lowest RNA binding activity of this series, although all of them retain the ability to fold into an alpha -helical conformation. On the other hand, Thr-24 mutation to alanine did not significantly affect the RNA binding capabilities. In contrast, Ile-33 was showed to be important, reflecting the importance of hydrophobic contacts between bulky amino acid side chains and RNA bases. The significance of this result increases if we consider that a bulky hydrophobic residue is present in a similar position in most of the carmovirus MPs characterized so far (Fig. 5A). This fact has been reported for other protein and peptide-RNA complexes. For instance, tryptophan and isoleucine residues have been identified as important amino acids to make specific contacts with the targeted RNA in peptides derived from the N-antitermination protein of phages lambda  (63) and P22 (64), respectively.

The overall results obtained in the present study suggest that the stabilization of nascent alpha -helical conformations in the unbound form of alpha -helical RNA-binding peptides, together with other relevant contributions, could have a role in decreasing the energy gap between the unbound and unfolded peptide and the fully folded peptide bound to RNA. Such a helix is identified in p7 in the segment 29AKDAIRK35. It is noteworthy that a similar motif could be found in CmPP16-1, the first plant protein described as a paralog of a viral MP that binds RNA with no sequence specificity (17) (Fig. 5B). This motif in CmPP16-1, which would have the potential to fold into an alpha -helix, is also preceded by positively charged residues, as in CarMV p7. This striking sequence similarity is even found between a region of CmPP16-1 and the conserved C terminus of CarMV p7 (Fig. 5B). In addition, the sequence-specific RNA-binding alpha -helical peptide from phage P22 N-antitermination protein (64) also shows a remarkable sequence similarity to p7 in this region (Fig. 5C). The phage peptide contains the isoleucine residue described as important for the p7-RNA interaction, the presence of the amino acid cluster Asn-Ala-Lys-Thr-Arg-Arg at the N terminus (as compared with the cluster Gln-Lys-Thr-Arg-Arg, in p717-35), and an aspartic acid residue in a positively charged context. Thus, it seems that sequence-specific RNA-binding domains, like the one from P22 N protein, and non-sequence-specific RNA-binding domains from CarMV p7 MP, despite their different origins and mechanisms of action, could share core structural elements that actively participate in the RNA binding.

Previous reports on nucleic acid binding peptides have suggested that the distribution of amino acids important for binding along the alpha -helical projection might be of significant importance. In this sense, the amino acids from an RNA-binding peptide derived from the HIV regulatory protein Rev that make base-specific contacts are distributed ~260° around the alpha -helix (65). When placed in an alpha -helical projection, the three consecutive amino acids from p717-35 important for RNA binding, namely Ile-33, Arg-34, and Lys-35, are distributed ~300° (Fig. 5D). By comparison, in the DNA-binding alpha -helix of GCN4, this class of amino acids are more tightly distributed to one face of the helix (~180° (66)). These observations could be related to the inherent structural flexibility of RNA when compared with DNA that, in turn, determines the structural requirements that have to be present in RNA- or DNA-binding proteins.

In conclusion, despite the relative paucity of structural data from RNA-protein complexes, some generalizations regarding the molecular mechanisms of RNA recognition can be made. In most cases examined to date, RNA recognition appears to occur via an "adaptive binding" mechanism wherein the RNA and the protein undergo significant conformational changes upon complex formation (19). The interaction between viral MPs and their target RNA would occur following such an adaptive mechanism as deduced from the data obtained in the present study. Thus, our structural studies of a peptide whose sequence corresponds to the RNA-binding domain of CarMV p7 demonstrate that the peptide populates a nascent alpha -helix in aqueous solution that is further stabilized in the presence of RNA or secondary structure inducers. Further structural studies with the whole p7 protein will shed more light on the RNA recognition process of this remarkable class of proteins.

    ACKNOWLEDGEMENT

We thank Alicia García for excellent technical work.

    FOOTNOTES

* This work was supported by European Union Biotechnology Grant BIO4-CT97-2086 (to E. P-P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ304989.

§ Recipient of a long term Ph.D. fellowship from the University of Valencia.

** To whom correspondence should be addressed: Dept. de Bioquímica i Biologia Molecular, Universitat de València, E-46100 Burjassot, València, Spain. Tel.: 34-963864868; Fax: 34-963864635; E-mail: paya@uv.es.

Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M100706200

2 M. C. Cañizares, J. F. Marcos, and V. Pallás, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: MP, movement protein; Fmoc, N-(9-fluorenyl)methoxycarbonyl; TMV, tobacco mosaic virus; CarMV, carnation mottle virus; TFE, trifluoroethanol; ssRNA, single-stranded RNA; ssDNA, single-stranded DNA; MOPS, 4-morpholinepropanesulfonic acid; EMSA, electrophoretic mobility shift assay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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