From the 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
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ABSTRACT |
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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 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 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 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 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 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.
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%
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
From these results, we conclude that the central region of p7, as
represented by p717-35, is an inducible 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.
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 RNA Binding Analysis by Alanine-substituted Peptides--
To
determine amino acid residues within the predicted
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
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 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 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
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 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 The overall results obtained in the present study suggest that
the stabilization of nascent Previous reports on nucleic acid binding peptides have suggested
that the distribution of amino acids important for binding along the
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 /
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
-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
-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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
-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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
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
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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").
-helical and
-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.
-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
-helical polypeptides (40) or submicellar concentrations
of SDS, which is considered a template that would stabilize both
-helical or
-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
-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
-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).
-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
-sheet that would account for the overall
-sheet content of p7.
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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.
-helical inducible protein motif.
There are many examples in the literature of RNA-binding peptides
and/or protein domains that fold in an
-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
-helical structure in p7 is
24TRRSVAKDAIRK35 (data not shown). In order to
confirm this putative
-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
-helical conformation is
characterized by upfield and downfield shifts of the
H and
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
-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
1H
and 13C
(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
-helix were found in water solution (d
N i, i + 3 between Ser-27 and Lys-30 and between Asp-31 and Arg-34;
d
N i, i + 4 between Asp-31 and Lys-35, data not shown) corresponding with strings
of dNN NOEs. Furthermore, additional
d
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 ( 1 H
)
and for 13C (
13C
) 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 -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
-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
-helix identified at the C terminus of
p717-35.
-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
-helical conformation.
All five basic residue substitution analogs retained propensities to
fold into an
-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
-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.
Amino acid sequence, RNA binding properties, and -helical content of
the alanine-scanning substitution analogs of p717-35
-helical conformation in
this polypeptide segment, as concluded from our NMR data (medium range
NOEs, d
N i,
i + 3 between Asp-31 and Arg-34;
d
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.
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-RNA-binding protein with an
estimation of 42%
-sheets, 10%
-helices, 5% turns, and 43%
random coil. This folding in mixed
and
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
-helices separated by
-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
-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
-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.
-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
-sheet secondary structure in aqueous solution that probably
accounts for the overall
-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
-sheet protein domain mediates the protein-protein interaction (57).
It is tempting to speculate that this conserved
-sheet domain could
be responsible for the protein-protein interactions that confer
cooperativity to the binding process of p7 to RNA (29).
-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
-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
-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).
-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
(63) and P22 (64), respectively.
-helical conformations in the unbound
form of
-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
-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
-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.
-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
-helix (65). When placed in an
-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
-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.
-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.
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