From the Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (SIC-UAM)), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
Received for publication, December 14, 2000, and in revised form, March 15, 2001
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ABSTRACT |
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The bacteriophage The The coiled-coil motif was first described as the main structural
element of a large class of eukaryotic fibrous proteins such as
tropomyosin (284 residues) and myosin (~1100 residues) (3). Structural analysis of in vitro assembled smooth muscle
myosin filaments revealed that they are flat side-polar sheets in which adjacent molecules overlap by ~14 nm. A noteworthy feature of this
assembly mechanism is that the packing is width-limiting but not
length-limiting (4). Coiled-coils are also the main structural elements
in the assembly of intermediate filament proteins (IFs),1 a superfamily of
10-nm fibers ubiquitous in multicellular eukaryotes (5). Despite the
rather low primary sequence identity, all the IFs have a long and
central Protein p1 from Bacillus subtilis phage In this report and as an approach to understanding the assembly
mechanism underlying the formation of p1 Bacterial Strains, Plasmids, and Growth
Conditions--
Escherichia coli XL1-Blue (Stratagene) was
used as host for the plasmids constructed in this work. Plasmid
pMalE-p1 Protein Purification and Factor Xa Protease
Cleavage--
Purification of maltose-binding protein (MalE) fusion
proteins has been described (12). Protein preparations were
concentrated using a Microcon microconcentrator 10 (Amicon). The
concentration of the MalE fusion proteins was measured by Lowry method
using bovine serum albumin as a standard. For digestion with protease factor Xa (New England Biolabs), protein preparations were diluted to
16 µM in dialysis buffer (20 mM Tris-HCl, pH
7.5, 200 mM NaCl, and 10 mM
N-terminal Sequencing of the Wild-type and Mutated p1 Electrospray Mass Spectrometry--
Analyses were performed with
a quadrupole Hewlett-Packard 1100 MSD mass spectrometer by using
an electrospray interface. MalE fusion proteins were treated with
factor Xa. The digested preparations were fractionated in an HPLC
apparatus (HP Series 1100) with an autosampler (injection volume 5 µl) equipped with a Zorbax 300 SB C18 column
(150 × 2.1 mm, 5-µm particle size). The mobile phase was a
mixture of solvent A (acetonitrile + 0.1% trifluoroacetic acid) and
solvent B (0.1% trifluoroacetic acid in water) according to a step
gradient over 40 min, changing from 85% B at 7 min to 5% B at 40 min
at a flow rate of 0.2 ml/min. Detection was accomplished by using a
diode array detection system Series 1100 (Hewlett-Packard), storing the
signal at a wavelength of 220 nm and 280 nm. A personal computer system
running Hewlett-Packard software was used for data acquisition and
processing. In the atmospheric pressure electrospray ionization method,
the eluted compounds were mixed with nitrogen in the heated nebulizer
interface, and polarity was tuned to positive. Adequate calibration of
electrospray ionization parameters (needle potential, gas temperature,
nebulizer pressure) was required to optimize the response and to obtain
a high sensitivity of the molecular ion. The selected values were:
needle potential, 4000 V; gas temperature, 310 °C; drying
gas, 10 ml/min; nebulizer pressure, 40 p.s.i.g. (1 p.s.i. = 6894.76 pascals). The fragmentor was jumped from 50 to 140 V for
different values of m/z.
Sedimentation through Glycerol Gradients--
Aliquots of the
protein preparations were loaded on to 5-ml glycerol gradient (15-30%
in dialysis buffer; see above) prepared in a Beckman polyallomer
centrifuge tube (13 × 51 mm). Centrifugation was performed at
62,000 rpm and 4 °C in a Beckman SW.65 rotor for the indicated time.
After centrifugation, ~170-µl fractions were collected by
puncturing the bottom of the tubes. The material sedimented at the
bottom of the tubes was resuspended in loading buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 5% Electron Microscopy--
Protein preparations were diluted to
0.5-1.5 µM in dialysis buffer (see above) and
immediately applied to carbon-coated copper grids for 2 min. Grids were
then washed with a few drops of water and stained for 30 s with
1% uranyl acetate. Electron micrographs were taken in a Jeol 1010 electron microscope at 80 kV.
Protein p1 Site-directed Mutagenesis of the Putative Coiled-coil of
p1 Characterization of the Wild-type and Mutated p1 Assembly of p1
We also performed kinetic experiments in which factor Xa was added to a
less concentrated MalE-p1 Residues Met53 and Leu60, but Not
Leu39, Are Essential for p1
The ability of protein p1
We have also studied the assembly properties of protein p1
Collectively, the above results demonstrate that residues
Met53 and Leu60, but not Leu39, of
protein p1 Replacement of Leu46 by Val in p1
The behavior of protein p1
The above results demonstrate that residue Leu46 of
p1 Conclusion--
Protein p1 has a short 29 replication protein
p1 self-interacts in vitro, generating highly ordered
structures. Specifically, the 53-amino acid protein p1
N33, which
retains the sequence of p1 spanning amino acids Met34 to
Lys85, assembles into two-dimensional protofilament sheets.
The region of protein p1 located between residues Glu38 and
Asn65 presumably forms an
-helical coiled-coil
structure. Here we have examined the role of this coiled-coil sequence
in the formation of protofilament sheets. Using sedimentation assays
and negative-stain electron microscopy analysis, we demonstrate that
residues Leu46, Met53, and Leu60,
but not Leu39, are essential for p1
N33 assembly into
sheets. Remarkably, replacement of Leu46 by Val shifts the
pathway of molecular assembly, leading to the formation of filamentous
polymers ~10 nm in diameter. These results show, for the first time,
that a short coiled-coil motif can mediate protein assembly into
protofilament sheet structures.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helical coiled-coil is probably the most widespread
subunit oligomerization motif found in proteins. Coiled-coils consist of two to five right-handed amphipathic
-helices that wind around each other to form a slightly left-handed supercoil (1, 2). The
formation of a coiled-coil is dependent primarily on the presence of
heptad repeat sequences of a form denoted
[abcdefg]n, where positions a and
d are mostly occupied by hydrophobic residues, and positions
e and g are frequently filled with charged residues.
-helical domain (~42 heptads), which is flanked by
nonhelical end domains. The
-helical domain has the ability to form
a parallel, unstaggered two-stranded coiled-coil. Formation of these
dimeric structures constitutes the first step in the assembly of
intermediate filaments, which proceeds further by different modes of
dimer-dimer interactions (6). Cytoplasmic IFs readily assemble in
vitro into ~10-nm wide filaments. These structures are similar
in appearance to those seen in cells (7). Unlike cytoplasmic IFs,
lamins, a class of nuclear IFs, are also able to assemble into
paracrystal structures under certain in vitro conditions (8,
9). In vivo, different lamin proteins form the nuclear
lamina, a thin fibrous structure immediately underlying the inner
nuclear membrane of most eukaryotic cell nuclei (10).
29 has the
capacity to polymerize in vitro. This small viral protein
(85 residues) presumably assembles in vivo into a structure
that associates with the bacterial membrane (11, 12). Recently, we have
proposed that the membrane-associated p1 structure functions as a
scaffold to which the
29 replisome becomes attached to initiate
viral DNA replication (13). Although the nature of p1 polymers in vivo is unknown, previous in vitro studies demonstrated
that the 53-amino acid protein p1
N33, which retains the sequence of
p1 spanning amino acids Met34 to Lys85,
assembles into large polymers that show a parallel array of longitudinal protofilaments. These structures are two-dimensional protofilament sheets whose length and width depends on the
polymerization time (12). According to computer algorithms, protein p1
as well as p1
N33 contains a short
-helical coiled-coil sequence
(~3 heptad repeats) (this work), suggesting that such a motif might be involved in the intermolecular interactions that lead to the formation of two-dimensional protofilament sheets. To our knowledge, coiled-coils as structural elements in the assembly of such structures have not been previously described. Negatively stained p1
N33 sheets
examined in the electron microscope resemble polymers formed under
particular in vitro conditions by FtsZ (14, 15), which forms
the cytoskeletal framework for cell division in all bacteria (16), and
by the
/
-tubulin heterodimer (17-19), which is the structural
subunit of the eukaryotic microtubules (20). However, unlike protein
p1
N33 (12), FtsZ (~40 kDa) and tubulin (~50 kDa each monomer)
lack a coiled-coil motif (21, 22). Furthermore, polymerization of both
proteins is regulated by GTP hydrolysis (23). Thus, the p1
N33
protofilament sheets must be structurally different from those formed
by FtsZ and tubulin.
N33 protofilament sheets, we
carried out kinetic experiments in which the p1
N33 assembly reaction
was followed by sedimentation assays and negative-stain electron
microscopy analysis. In addition, we performed a mutational analysis to
examine whether the
-helical coiled-coil sequence present in protein
p1
N33 is involved in the formation of sheet structures.
Specifically, amino acid residues occupying the d position
of the heptad repeats were replaced by other apolar amino acids. The
results obtained indicate that a nucleation-elongation mechanism
mediated by a coiled-coil sequence is involved in the formation of
p1
N33 sheets. Furthermore, we show that a single conservative
substitution within the hydrophobic core of such a coiled-coil motif is
sufficient to shift the pathway of protein assembly from
two-dimensional protofilament sheets to ~10-nm-wide filaments.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
N33 has been previously described (12). It is based on the
pMAL-c2 expression vector (New England Biolabs). To introduce
single-amino acid substitutions in protein p1
N33, a two-step
mutagenesis method based on the polymerase chain reaction was used. In
the first step, two reactions (A and B) were carried out using plasmid
pMalE-p1
N33 as template. Reaction A contained as primers the
oligonucleotide HindIII (5'-CGACG GCCAGTGCCAAGCTTG-3') and
the oligonucleotide I, which carries the modified codon (L39A: 5'-ACCT
TGAGGCAGAAAAGAAGATG-3'; L46A: 5'-GATGACTAAAGCAGAGCATGAA-3'; L46V:
5'GATGACTAAAGTAGAGCATGAA-3'; M53A:
5'-ATAAACTCGCGAAAAACGCATTG-3'; L60I:
5'-TTGTATGAGATTTCTAGGATG-3'). Reaction B contained as
primers the oligonucleotide SacI (5'-CGATGAAGCCCTGAAAGAC-3') and the oligonucleotide II, whose sequence is complementary to oligonucleotide I. In the second step of the mutagenesis procedure, the
polymerase chain reaction fragments obtained in the reactions A and B
were mixed and used as template in a polymerase chain reaction that
used oligonucleotides HindIII and SacI (see
above) as primers. The resulting polymerase chain reaction fragments, digested with HindIII and SacI, were used to
replace the HindIII-SacI region of plasmid
pMalE-p1
N33. In the mutant plasmids, as in the wild-type plasmid
pMalE-p1
N33, there is a Gly codon between the
Ile-Glu-Gly-Arg-encoding sequence and the Met34 codon of
gene 1. Protease factor Xa cleaves after the sequence Ile-Glu-Gly-Arg.
The nucleotide sequence of the inserts was confirmed by DNA sequencing
using the dideoxy nucleotide chain termination method (24).
Plasmid-containing cells were grown in LB broth (25) supplemented with
ampicillin (100 µg/ml).
-mercaptoethanol). Factor Xa was added to the protein preparation at
a ratio of 1.25% (w/w) the amount of fusion protein. The reaction was
incubated at room temperature for the indicated time. The cleavage with
factor Xa was monitored by SDS-Tricine-PAGE (26). The percentage of
uncleaved fusion protein was calculated by densitometric scanning of
the gel in a Molecular Dynamics 300A densitometer.
N33
Proteins by Edman Degradation--
MalE fusion proteins were treated
with protease factor Xa. The digested protein preparations were loaded
onto an SDS-Tricine polyacrylamide gel. After electrophoresis, the
p1
N33 products were transferred electrophoretically to Immobilon-P
membranes (Millipore) using a Mini Trans Blot (Bio-Rad) at 100 mA and
4 °C for 60 min. N-terminal sequencing was performed on an Applied Biosystems 473A protein sequencer.
-mercaptoethanol, and
30% glycerol). Aliquots from each fraction were analyzed by SDS-Tricine-PAGE (26).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
N33 Has an
-Helical Coiled-coil Motif--
Protein
p1
N33 is an N-terminal-truncated p1 protein that retains the
sequence spanning amino acids Met34 to Lys85.
This 53-amino acid protein has Gly as first residue (Fig.
1A). In solution, protein
p1
N33 assembles into two-dimensional protofilament sheets (Fig.
1B) (12). Polymerization of p1
N33 into sheets takes place
in the absence of auxiliary factors, suggesting that the information
necessary to form such polymers relies upon its structure. According to
the PHD secondary structure prediction program (27), the region of
protein p1 located between residues Ile35 and
Arg62 has a high probability (0.5-1) of adopting an
-helical conformation. Moreover, the COILS prediction program (1)
revealed that the region of p1 spanning amino acids Glu38
to Asn65 has a high probability (0.98) of forming an
-helical coiled-coil structure (Fig. 1A). This structure
is characterized by a repeating heptad motif (a to
g), with the amino acid side chains being predominantly
hydrophobic at the a and d positions of each
heptad and hydrophilic elsewhere (1). Since each heptad repeat forms
two
-helical turns, the a and d residues are
located on the same face of the helix forming a hydrophobic core (Fig.
1C).
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Fig. 1.
Relevant features of
p1 N33, a truncated p1 protein that lacks the
N-terminal 33 amino acids. A, amino acid sequence of
the 53-amino acid protein p1
N33. This protein has Gly as the first
residue. It retains the region of p1 spanning amino acids
Met34 to Lys85. The amino acid position refers
to the p1 sequence (12). The predicted
-helical coiled-coil sequence
is indicated. Heptad repeats are denoted
[abcdefg]n. Residues at the
d positions were changed to Ala, Val, or Ile, as indicated.
B, electron micrograph of a negatively stained p1
N33
protofilament sheet (12). The scale bar represents 50 nm.
C, helical wheel projection of residues Leu39 to
Met63 of the p1
N33 sequence. Residues at position
d are boxed.
N33--
It has been shown that interhelical hydrophobic
interactions are a dominant factor in the stabilization of coiled-coils
(28). Furthermore, a systematic mutational analysis in the yeast
transcription factor GCN4 revealed that conservative substitutions
targeting the hydrophobic residues at positions a and
d alter the packing and stoichiometry of coiled-coils (29).
To determine whether assembly of protein p1
N33 (referred to as
wild-type protein) into protofilament sheets is mediated by a
coiled-coil sequence, we introduced single substitutions at the
four d positions: Leu39,
Leu46, Met53, and Leu60. These
residues were changed to Ala, Val, or Ile, as shown in Fig.
1A. The changes were designed taking into account
coiled-coil predictions (1) and general suggestions for conservative
substitutions (30).
N33
Proteins--
Like the wild-type protein, the mutated p1
N33
proteins (L39A, L46A, L46V, M53A, and L60I) were fused to the
maltose-binding protein (MalE) to facilitate their purification (see
"Experimental Procedures"). The purified MalE fusion proteins,
concentrated to 16 µM, were further treated with protease
factor Xa. This protease cleaves after the sequence IEGR, which
separates MalE from p1
N33. SDS-Tricine-PAGE analysis of digested
MalE-p1
N33 preparations showed that only two products with the
mobility expected for MalE (42.4 kDa) and p1
N33 (6.1 kDa) were
generated (Fig. 2A). The same
result was obtained when the mutated MalE fusion proteins where cleaved
with factor Xa (not shown). Furthermore, to determine accurately the
molecular mass of the wild-type and mutated p1
N33 products, digested
MalE fusion proteins were fractionated by HPLC. Two fractions, which
corresponded to the MalE and p1
N33 monomeric forms, were obtained.
Each fraction was analyzed by electrospray-mass spectrometry. As shown
in Table I, the molecular mass of the p1
N33 products differed from the expected value by 3.37-6.24 Da. In
all the cases, the measured molecular mass of the MalE component was
42476.48 ± 6.93 Da. These results, together with N-terminal
sequencing of the wild-type and mutated p1
N33 proteins (not shown),
demonstrate that factor Xa cleavage occurs exclusively at the specific
site.
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Fig. 2.
Treatment of
MalE-p1 N33 with protease factor Xa.
A, SDS-Tricine-PAGE analysis of a MalE-p1
N33 preparation
(16 µM) digested with factor Xa. The molecular weight of
prestained proteins (Life Technologies, Inc.) used as markers is
indicated in kilodaltons. B, factor Xa was added to a
MalE-p1
N33 protein preparation (16 µM) at a ratio of
1.25% (w/w) of the amount of fusion protein. The reaction mixture was
incubated at room temperature. At the indicated times, samples were
analyzed by SDS-Tricine-PAGE. The gel was stained with Coomassie Blue.
The concentration of p1
N33 at different times of addition of factor
Xa was determined by calculating the percentage of uncleaved
MalE-p1
N33 by densitometric scanning of the gel.
Comparison of expected and measured molecular masses (Da) of the
wild-type and mutated p1N33 proteins
N33 into Protofilament Sheets Can Be Envisaged as
a Two-step Process--
Sedimentation assays through glycerol
gradients showed that MalE-p1
N33 (48.5 kDa) sedimented at the
position of 88 kDa (Table II). Therefore,
this fusion protein is likely a dimer throughout the concentration
range analyzed (1.5-16 µM). Previous studies demonstrated that, after adding factor Xa to a MalE-p1
N33
preparation, protein p1
N33 self-associated into protofilament sheets
(Fig. 1B), whereas MalE remained in solution as monomer
(12). These findings reveal that MalE, when fused to p1
N33, provides
a steric hindrance for p1
N33 assembly. This feature has been crucial
in studying the assembly properties of protein p1
N33 in solution. To
this end, factor Xa was added to a MalE-p1
N33 protein preparation (16 µM). At different times, samples of the reaction
mixture were analyzed by SDS-Tricine-PAGE (Fig. 2B). This
analysis allowed us to determine the percentage of uncleaved
MalE-p1
N33 and, thus, to estimate the concentration of protein
p1
N33 in the reaction. The partially digested samples were also
analyzed by sedimentation through glycerol gradients (Fig.
3) and negative-stain electron microscopy. After 4 h of factor Xa addition, when the
concentration of p1
N33 was 6 µM, p1
N33 complexes
sedimenting as the native MalE protein were detected (Fig.
3A). The same sedimentation position was observed at 8 h, when the p1
N33 concentration was 9 µM. Such a
position corresponded to 45 kDa, as determined by increasing the
centrifugation time of the sample in the presence of different marker
proteins (Fig. 3B). The MalE-p1
N33 molecules still
present in the preparation sedimented as 88 kDa (Table II). At these
times, negatively stained specimens were not visualized in the electron microscope. These results indicated that, after removal of MalE by
cleavage with protease factor Xa, protein p1
N33 was able to self-associate into small oligomers, which were accumulated.
Interestingly, at 19 h, when the concentration of p1
N33 was 14 µM, the amount of p1
N33 complexes sedimenting at the
position of 45 kDa decreased, and p1
N33 complexes migrating faster
appeared (Fig. 3A). Moreover, the amount of protein p1
N33
recovered at the bottom of the glycerol gradient increased (Fig.
3C). Hence, assembly of p1
N33 into larger structures was
taking place. Negative-stain electron microscopy analysis of this
preparation showed that protofilament sheets (Fig. 1B) with
different widths (14-62 nm) were formed. From these results we
conclude that assembly of p1
N33 into protofilament sheets can be
envisaged as a two-step process: (i) a nucleation step that leads to
the accumulation of small oligomers, and (ii) an elongation step in
which these oligomers are assembly intermediates able to generate
protofilament sheets.
Sedimentation position of the MalE fusion proteins in glycerol
gradients
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Fig. 3.
Assembly of p1 N33
into sheets. Partially digested MalE-p1
N33 samples from the
reaction mixture shown in Fig. 2B were analyzed by
sedimentation through glycerol gradients (15-30%). A,
samples taken at the indicated times were subjected to centrifugation
for 11 h, as indicated in "Experimental Procedures."
Sedimentation is from right to left. After fractionation of the
glycerol gradients, aliquots from each fraction were analyzed by
SDS-Tricine-PAGE. Densitometric scanning of the gel stained with
Coomassie Blue was used to determine the amount of MalE and p1
N33 in
each fraction (arbitrary units). B, a sample taken at 8 h, when the p1
N33 concentration was 9 µM, was also
centrifuged for 20 h in the presence of different marker proteins
(open boxes): alcohol dehydrogenase (150 kDa), bovine serum
albumin (67 kDa), and
29 single-stranded DNA-binding protein (13.3 kDa). After fractionation of the glycerol gradient, aliquots from each
fraction were analyzed by SDS-Tricine-PAGE. The fraction at which the
maximal amount of each protein appeared was determined. The maximal
amount of MalE was detected in the same fraction as the maximal amount
of p1
N33. C, samples taken at the indicated times were
subjected to centrifugation for 11 h, as shown in panel
A. After fractionation of the glycerol gradients, the material
sedimented at the bottom of the tubes was resuspended in loading buffer
(60 mM Tris-HCl, pH 6.8, 2% SDS, 5%
-mercaptoethanol,
and 30% glycerol). Equivalent volumes were analyzed by
SDS-Tricine-PAGE. The gel was stained with Coomassie Blue.
N33 protein preparation (4 µM). In this case, at 48 h of addition of factor Xa,
when the concentration of p1
N33 was 3.5 µM, negatively
stained sheets were visualized by electron microscopy (not shown).
Therefore, p1
N33 polymerization is a function not only of protein
concentration but also of time.
N33 Assembly into
Protofilament Sheets--
We next investigated whether residues
Met53 and Leu60 of p1
N33 are necessary for
assembly into protofilament sheets (Fig. 1). To this end, we
constructed the MalE-p1
N33.M53A and MalE-p1
N33.L60I fusion
proteins, in which residues Met53 and Leu60 of
p1
N33 were replaced by Ala and Ile, respectively. In contrast to
MalE-p1
N33, the MalE-p1
N33.M53A fusion protein sedimented in
glycerol gradients at the position of 52 kDa, which may correspond to
the monomeric form (Table II). At 8 h of addition of factor Xa to
a MalE-p1
N33.M53A protein preparation (16 µM), the
p1
N33.M53A concentration was 12 µM (Fig.
4A). It was measured as
described for the wild-type protein (Fig. 2B). At this time,
p1
N33.M53A sedimented at the position of 12 kDa, whereas MalE
sedimented as 45 kDa. Sedimentation of p1
N33.M53A at such a position
was also observed at later times of factor Xa addition (not shown). Furthermore, protein complexes migrating faster were not detected, and
negatively stained structures were not visualized. These results indicate that protein p1
N33.M53A cannot generate highly ordered structures.
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Fig. 4.
Sedimentation studies of protein
p1 N33.M53A (A) and
protein p1
N33.L60I (B).
The corresponding MalE fusion protein (16 µM) was treated
with factor Xa for 8 h. At this time, when the concentration of
p1
N33.M53A and p1
N33.L60I was 12 µM and 14 µM, respectively, the reaction mixtures were loaded on to
a 15-30% glycerol gradient and subjected to centrifugation for
20 h. As markers, bovine serum albumin (BSA; 67 kDa)
and
29 single-stranded DNA-binding protein (SSB; 13.3 kDa) were loaded in the same gradient. After fractionation, aliquots
from each fraction were analyzed by SDS-Tricine-PAGE. Densitometric
scanning of the gels stained with Coomassie Blue was used to determine
the amount of the different proteins in each fraction (arbitrary
units). The native MalE protein sedimented at the position of 45 kDa.
The arrows indicate fractions at which the maximal amount of
each marker was detected.
N33 to form sheets was also affected when
residue Leu60 was changed to Ile. Although the
MalE-p1
N33.L60I fusion protein behaved in solution as the wild-type
protein (Table II), the assembly properties of p1
N33.L60I, after
being cleaved from MalE, were different. As shown in Fig.
4B, at 8 h of addition of factor Xa to a
MalE-p1
N33.L60I preparation (16 µM), the p1
N33.L60I
concentration was 14 µM. At this time, protein
p1
N33.L60I sedimented at the position of 25 kDa. This position did
not change as a function of time (not shown). Moreover, negatively
stained specimens were not visualized. Thus, small p1
N33.L60I
oligomeric structures are formed, but they cannot assemble further into
larger arrays.
N33.L39A,
in which residue Leu39 was changed to Ala (Fig.
1A). Like MalE-p1
N33, protein MalE-p1
N33.L39A behaved
in solution as a dimer (Table II). Furthermore, after being cleaved
from MalE, protein p1
N33.L39A was able to generate protofilament
sheets (Fig. 6) following the same assembly pathway than the wild-type
protein (Fig. 3).
N33 are involved in the formation of sheets. Specifically, the change of Met53 to Ala as well as the change of
Leu60 to Ile generates structures that are not able to
function as assembly intermediates.
N33 Leads to the
Formation of 10-nm Filamentous Structures--
To find out whether
residue Leu46 of protein p1
N33 is essential for assembly
into protofilament sheets, we changed this amino acid residue to Val
(protein p1
N33.L46V) or Ala (protein p1
N33.L46A) (Fig.
1A). These mutated proteins were fused to MalE.
Sedimentation analysis through glycerol gradients showed that both
fusion proteins sedimented slightly faster than the wild-type
MalE-p1
N33 protein (Table II). We next examined the assembly
properties of p1
N33.L46V and p1
N33.L46A after being cleaved from
MalE by sedimentation assays (Fig. 5) and
negative-stain electron microscopy analysis (Fig.
6). At 4 h of addition of factor Xa
to a MalE-p1
N33.L46V protein preparation (16 µM), the
p1
N33.L46V concentration was 7 µM (Fig.
5A). It was measured as described for the wild-type protein
(Fig. 2B). At this time, p1
N33.L46V complexes migrating faster than MalE were detected. At 8 h of addition of factor Xa, samples of the reaction mixture were subjected to centrifugation for
11 h (Fig. 5A) or for 20 h in the presence of
protein markers (Fig. 5B). At this time, most of the
p1
N33.L46V complexes sedimented at the position of 115 kDa, although
complexes migrating more slowly were also observed (Fig.
5A). After 19 h of factor Xa addition, the
concentration of p1
N33.L46V was 14 µM. At this time,
the amount of complexes sedimenting at the position of 115 kDa was lower (Fig. 5A). This decrease correlated with an increase
in the amount of protein p1
N33.L46V recovered at the bottom of the glycerol gradient (Fig. 5C). Therefore, assembly of larger
structures was taking place. Analysis of this protein preparation by
negative-stain electron microscopy showed that long filamentous
polymers were formed (Fig. 6). These filaments had different lengths
and were ~10 nm in diameter.
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Fig. 5.
Sedimentation studies of
p1 N33.L46V and
p1
N33.L46A. A, factor Xa was
added to the corresponding MalE fusion protein (16 µM) at
a ratio of 1.25% (w/w) the amount of fusion protein. At different
times, samples of the reaction mixture were analyzed by
SDS-Tricine-PAGE (not shown) to determine the concentration of the
mutated p1
N33 protein, as indicated in the legend to Fig.
2B. Such samples were also analyzed by sedimentation through
glycerol gradients (15-30%). Centrifugation time was 11 h. After
fractionation of the gradients, the amount of the different proteins in
each fraction (arbitrary units) was calculated by densitometric
scanning of SDS-Tricine polyacrylamide gels stained with Coomassie
Blue. B, a MalE-p1
N33.L46V preparation (16 µM) was digested with factor Xa for 8 h. Then the
reaction mixture was subjected to centrifugation in a 15-30% glycerol
gradient for 20 h. As markers (open boxes), alcohol
dehydrogenase (150 kDa), bovine serum albumin (67 kDa), and
29
single-stranded DNA-binding protein (13.3 kDa) were used. The fraction
at which the maximal amount of each protein appeared was determined.
Native MalE sedimented at the position of 45 kDa (not shown).
C, a MalE-p1
N33.L46V preparation (16 µM)
was digested with factor Xa. At the indicated times, samples were
subjected to centrifugation in glycerol gradients for 11 h, as
shown in panel A. After fractionation, the material
sedimented at the bottom of the tubes was resuspended in loading
buffer. Equivalent volumes were analyzed by SDS-Tricine-PAGE. The gel
was stained with Coomassie Blue.
View larger version (101K):
[in a new window]
Fig. 6.
Electron micrographs of
p1 N33.L39A and
p1
N33.L46V structures. Protease factor Xa
was added to the corresponding MalE fusion protein (16 µM). At 19 h, samples were diluted and treated for
negative staining. Left, protofilament sheet formed by
protein p1
N33.L39A. When attached to the carbon, sheets tend to fold
over, indicating that both sides are identical. Sheets with different
widths were visualized. These structures were identical to the sheets
formed by the wild-type protein. Right, filamentous
structures formed by p1
N33.L46V. Although filaments of various
lengths were visualized, they were always ~10 nm wide. The
upper panel shows two filaments crossing each other. The
scale bars represent 100 nm.
N33.L46A in solution was different (Fig.
5A). As in the case of p1
N33.L46V, p1
N33.L46A
complexes sedimenting at the position of 115 kDa were observed at 4 and 8 h of factor Xa addition, when the concentration of p1
N33.L46A was 8 and 14 µM, respectively. However, in contrast to
p1
N33.L46V, the amount of protein detected at such a position did
not decrease at 19 h (Fig. 5A). Moreover, neither
protein p1
N33.L46A was found at the bottom of the glycerol gradient
nor were negatively stained specimens visualized (not shown). Thus,
protein p1
N33.L46A is deficient in the formation of
higher-ordered structures.
N33 is essential for assembly into protofilament sheets.
Specifically, replacement of Leu46 by the hydrophobic amino
acid Val is sufficient to change the pathway of molecular assembly,
generating 10-nm filamentous structures. These structures seem to be
constituted by a discrete number of protofilaments adopting a helical
array. Like formation of p1
N33 sheets, assembly of p1
N33.L46V
into filaments can be envisaged as a two-step process. During the
nucleation step, oligomeric structures are accumulated that show a
sedimentation behavior different from the p1
N33 assembly
intermediates. Furthermore, replacement of Leu46 by Ala
results in a protein that can self-interact into oligomeric structures.
These oligomers show a sedimentation behavior similar to the
p1
N33.L46V assembly intermediates. However, despite this similarity,
they must be structurally different because the p1
N33.L46A oligomers
cannot assemble further into larger arrays.
-helical coiled-coil
sequence located between Glu38 and Asn65 (~3
heptad repeats). This motif is also present in p1
N33, an N-terminal-truncated p1 protein that self-associates in
vitro into two-dimensional protofilament sheets. We show in this
study that assembly of the 53-amino acid protein p1
N33 into sheets can be envisaged as a two-step process. First, a nucleation step leads
to the accumulation of small oligomeric structures. And second, these
structures are able to function as assembly intermediates. In addition,
we demonstrate that residues Leu46, Met53, and
Leu60 of p1
N33 are essential for assembly into sheets.
These amino acid residues are located at the hydrophobic d
position of the heptad repeats. Interestingly, substitution of residue Leu46 with the smaller hydrophobic amino acid Val allows
protein p1
N33 to assemble into long filaments, whose diameters are
~10 nm. These findings are of particular relevance because they show
for the first time that (i) a short coiled-coil motif functions to
assemble a small protein into two-dimensional protofilament sheets and (ii) a single conservative substitution targeting the hydrophobic core
of the coiled-coil sequence is sufficient to change the pathway of
molecular assembly from two-dimensional sheets to 10-nm filamentous structures.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to Dr. M. Espinosa for critical reading of the manuscript, Dr. A. Abril for helpful comments, and M. J. Vicente for help with electrospray-mass spectrometry experiments.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant 2R01 GM27242-21, Dirección General de Investigación Científica y Técnica Grant PB98-0645, and by the European Union Grants BIO4-CT98-0250 and ERBFMX-CT97-0125. The institutional help of Fundación Ramón Areces is acknowledged.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.
Supported by the Ministerio de Educación y Cultura (Spain).
§ Recipient of a predoctoral fellowship from Ministerio de Ciencia y Tecnología (Spain).
¶ To whom correspondence should be addressed. Tel.: 34-91-3978435; Fax: 34-91-3978490; E-mail: msalas@cbm.uam.es.
Published, JBC Papers in Press, March 29, 2001, DOI 10.1074/jbc.M011296200
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ABBREVIATIONS |
---|
The abbreviations used are: IF, intermediate filament protein; MalE, maltose-binding protein; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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