From the Department of Biochemistry and Molecular
Biology and the § Department of Chemistry, University of
Massachusetts, Amherst, Massachussets 01003
Received for publication, December 11, 2000, and in revised form, February 13, 2001
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ABSTRACT |
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The signal recognition particle (SRP) is an
RNA-protein complex that directs ribosomes to the rough endoplasmic
reticulum membrane by binding to targeting signals found on the nascent chain of proteins destined for export to the endoplasmic reticulum. We
found evidence from studies with fragments of the protein component of
the Escherichia coli SRP that a long hydrophobic loop (the so-called "finger loop") is detrimental to the stability of its signal peptide-binding domain, the M domain. This hydrophobic loop is
highly conserved and thus may have a critical role in the function of
the SRP. Given our previously reported evidence that 4.5 S RNA
stabilizes the tertiary fold of the M domain (Zheng, N., and Gierasch,
L. M. (1997) Mol. Cell 1, 79-87), we now propose that
the functional requirement for 4.5 S RNA resides in its ability to
counteract the destabilizing influence of the finger loop.
In eukaryotic cells the signal recognition particle
(SRP)1 targets ribosomes
synthesizing secretory and membrane proteins to the rough endoplasmic
reticulum membrane. The Escherichia coli SRP is composed of
a single protein subunit, termed Ffh, which binds to a 114-nucleotide
RNA molecule, 4.5 S RNA. The E. coli SRP has been shown to
direct nascent membrane proteins into the inner membrane and therefore
bears a strong functional resemblance to its counterparts in eukaryotic
organisms (1-4). Limited proteolysis experiments define two domains in
Ffh termed the "NG" and "M" domains (5). The C-terminal M
domain contains the RNA-binding site and can be cross-linked to signal
sequences (6). The NG domain contains a Ras-like GTPase domain and a
novel small helical N-domain (7). The crystal structure of
Thermus aquaticus Ffh shows the M domain to consist of four
As yet there is no clear explanation why RNA is necessary for SRP
function (9). Based on the recent crystal structure of the M domain-4.5
S RNA complex, it has been proposed that RNA interacts directly with
the signal peptide (10). On the other hand, our previous studies with
E. coli Ffh point to an important structural role for RNA
(11). Our circular dichroism and proteolysis experiments showed that
4.5 S RNA stabilizes the tertiary fold of the M domain, which is highly
flexible in the absence of 4.5 S RNA. Indeed the recent observation
that RNA catalyzes the association of Ffh with its receptor has been
attributed to possible RNA-induced conformational changes in the
protein (12). We now report that the metastability of the E. coli Ffh M domain in the absence of RNA can be attributed to the
presence of the long, hydrophobic finger loop. This loop is conserved
in all homologues of SRP and thus may have a critical role in the
function of the SRP. In turn, we propose that RNA is functionally
necessary to counter the destabilizing effect associated with this loop.
Protein and RNA Preparation--
Ffh and 4.5 S RNA were prepared
as described previously (11). The M domain (residues
Gly326-Met435), amino deletion mutant
(residues Lys365-Met435) and finger loop
deletion mutant (from Gly326-Met435 with
Gln340-Asp370 replaced by a GAGG linker
sequence) were overexpressed from pET20b vectors (Novagen) in
BL21DE3pLysS (Stratagene) cells and purified under denaturing
conditions by cation exchange chromatography using CM-Sepharose CL-6B
(Sigma) resin. Removal of denaturant by dialysis against water was used
to refold all of these proteins. To prepare the Lys324 Ffh
truncation mutant, a stop codon was introduced into the Ffh expression
plasmid at the Lys325 codon by site-directed mutagenesis
using the Stratagene QuikChange site-directed mutagenesis protocol. To
prepare the finger loop deletion mutant, a polymerase chain reaction
was used to introduce a unique NarI restriction site and two
Gly codons into the M domain expression plasmid upstream of the
Lys371 codon (via the 5'-primer) and to introduce a second
NarI site downstream of the Arg339 codon (via
the 3'-primer). The linear polymerase chain reaction product was then
cut with NarI, purified, and circularized with DNA
ligase. The identities of the M domain, amino deletion mutant, and finger loop deletion mutant were verified by matrix-assisted laser
desorption ionization mass spectrometry (see Fig. 3A
for a schematic description of these constructs).
Limited Proteolysis--
Ffh (1 mg
ml Circular Dichroism--
All CD data were acquired in 10 mM potassium phosphate buffer (pH 6.5) in a 2-mm path
length cell with protein concentrations of 5-10 µM. For
thermal unfolding experiments, the sample was heated at a rate of
20 °C h Gel Shift Assay--
0.2 µM 4.5 S RNA was mixed
with various concentrations of Ffh and M domain in buffer A
supplemented with 10% glycerol. 10-µl binding reactions
were supplemented with 3 µg of bakers' yeast tRNA (Sigma) per
binding reaction to reduce nonspecific binding. For competition binding
reactions, all components were thoroughly premixed before adding the
4.5 S RNA. The binding reactions were separated on 7% polyacrylamide
gels as described by Lentzen et al. (13).
Destabilizing Influence of the Finger Loop--
Crystal structures
of the E. coli Ffh M domain-4.5 S RNA complex (10) and of
the homologous M domain from the mammalian SRP (14) both revealed
lengthy loops equivalent to the T. aquaticus Ffh finger
loop. Moreover, sequence comparisons among homologues showed the size
and character of this loop to be largely conserved (Fig.
1B). Using limited
proteolysis, we found evidence that this disordered, exposed loop is
retained as a feature of the E. coli Ffh M domain in
solution. Elastase digestion of Ffh in the presence of 4.5 S RNA
generates an 8-kDa truncated fragment of the M domain which starts, by
N-terminal sequence analysis, at Lys365 (Fig.
2). Based on its approximate size on the
SDS-PAGE and on analysis of the digest by matrix-assisted laser
desorption ionization mass spectrometry, we concluded that this
elastase-resistant fragment corresponds with
Lys365-Met435. Thus, the protease cleaves
within the finger loop and leaves the region that encompasses helices
2-4 intact in the presence of 4.5 S RNA. This protease-stable core of
the M domain is marked in magenta on the structure in Fig.
1A.
In our subsequent analysis of recombinant versions of the elastase
fragment (Lys365-Met435, termed here the
"amino deletion mutant"; see Fig.
3A) and the M domain (residues
Gly326-Met435, see Fig. 3A), we
found that the finger loop has a markedly destabilizing effect on the
fold of the M domain. CD spectra of the amino deletion mutant and the M
domain both reveal a substantial amount of
Remarkably, the smaller amino deletion mutant had a considerably more
cooperative thermal unfolding profile than the fully intact M domain.
Its 222 nm ellipticity signal changed in a sigmoidal manner between 4 and 80 °C, with the transition fully reversible. These combined
structural data therefore revealed, quite unexpectedly, that the
truncated amino deletion mutant has a higher propensity to form a
tertiary structure than the fully intact M domain.
The unexpected gain in structural stability upon removing helix 1 and
the finger loop to form the amino deletion mutant suggests that the
unusually long hydrophobic finger loop (amino acids
Leu338-Asp370) destabilizes the folding of the
M domain. A contribution from helix 1 cannot be excluded, however. To
analyze the effect of the finger loop more directly, we prepared an
additional construct in which finger loop residues
Gln340-Asp370 were replaced with a flexible
GAGG linker sequence. Thirty-one residues in the loop, including the
conserved hydrophobic residues, were thus removed, creating the finger
loop deletion mutant (Fig. 3A). Strikingly, the protein
remained highly Solvent Accessibility of the Finger Loop within the Context of
Intact Ffh--
We surmised that the destabilizing effect associated
with the loop might be due to the exposure of its many hydrophobic
residues on the protein surface. However, whether the loop is
surface-exposed when the protein is in solution is left uncertain by
the way it is involved in extensive intermolecular packing interactions
in the crystal structures of both T. aquaticus Ffh (8) and
Homo sapiens Ffh M domain (14). The facile elastase cleavage
we observe within the finger loop region (Fig. 2) suggested that this
loop is exposed on the surface of the M domain in solution. Milder conditions biased toward only one protease cut per molecule were used
in further experiments to probe whether the loop is surface-exposed within intact Ffh, prior to cleavage of the NG-M domain linker. Ffh
bearing an N-terminal dodecahistidine tag was digested with a low
concentration of elastase, and N-terminal fragments were identified by
monitoring for fragments that bound to Ni-NTA resin.
Two N-terminal fragments were identified that can be assigned as the
products of cleavage after Lys325, the starting point of
the M domain, by virtue of their slower electrophoretic mobility in
comparison with that of a mutant truncated at Lys324 (Fig.
5A, fragments labeled
A and B), and by the fact that they bind both to
Ni-NTA resin and to an anti-His tag antibody in Western blots (data not
shown). The cleavage region can be further narrowed down to between
Lys325 and Lys365 because proteolysis under
harsher conditions (Fig. 2) revealed that the
Lys365-Met435 region is highly stable to
proteolytic cleavage in the presence of RNA. Given that the bulk of the
Lys325-Lys365 region encompasses the finger
loop and that limited proteolysis generally cleaves flexible loops with
much higher propensity than regular secondary structure elements (15),
we conclude that the cleavages occur within the finger loop.
Furthermore, because fragments A and B retained the linker between the
NG and M domains, it follows that cleavage of the NG-M domain linker is
not a prerequisite for the finger loop to become protease-accessible.
These results therefore strongly argue that the finger loop is
solvent-exposed within the context of intact Ffh.
Intriguingly, analysis of the band intensities revealed an increase in
the amount of fragment A when the digestion of Ffh is conducted under
identical conditions in the presence of 4.5 S RNA (Fig. 5B,
lanes 2 and 3). Complicating the interpretation of these data is the difficulty of quantitating the amount of fragment
A in a way that accounts for any differences in gel loading or
proteolysis efficiency between lanes. However, this apparent increase
was observed consistently after conducting several parallel proteolysis
reactions under identical conditions in the presence and absence of 4.5 S RNA. Any increase in the exposure of the finger loop to the protease
must be induced in the intact protein by the RNA prior to cleavage. The
alternative possibility, that the RNA protects fragment A from further
cleavages, is ruled out because the product lacks the bulk of the
RNA-binding region and thus should not bind RNA. It is thus possible
that RNA binding drives the unfavorable exposure of the loop on the
protein surface. In addition, clear changes in the pattern of 27-37
kDa-sized fragments in the presence of RNA further indicate significant
RNA-induced structural changes in Ffh (Fig. 5B).
Previously we found that the Ffh M domain lacks a stable tertiary
structure as an isolated fragment in the absence of 4.5 S RNA (11). We
can now reconcile this finding with the presence of the finger loop,
which is shown here to destabilize the fold of the M domain. This
result may be explained by the nonpolar character of the loop, which
contains a number of highly conserved hydrophobic residues (Fig. 1,
A and B). Inspection of the crystal structure of
T. aquaticus Ffh (8) revealed that these conserved hydrophobic residues neither pack into the hydrophobic core of the M
domain nor interact with the NG domain but are instead buried by
packing interactions between molecules in the crystal lattice. Similar
packing interactions are observed in the crystal structure of the
homologous M domain from H. sapiens SRP (14). In both cases
the finger loop on one molecule interacts with the proposed signal
sequence binding groove on a neighboring molecule, thus satisfying the
need of both molecules to sequester hydrophobic residues. These two
crystal structures therefore provide evidence that the finger loop has
a preference for hydrophobic environments and cannot easily accommodate
its hydrophobic residues intramolecularly within the hydrophobic core
of the M domain.
Notably, however, neither these crystal structures nor the recent
structure of E. coli M domain bound to a fragment of 4.5 S
RNA (10) provided a picture of the finger loop interactions when the M
domain is in the context of soluble, intact Ffh. Our proteolysis data
have provided evidence that the finger loop, despite its
hydrophobicity, is exposed on the surface of the Ffh protein. Two
protease-susceptible regions were identified that can be mapped to a
portion of the M domain sequence dominated by the finger loop.
Therefore, any destabilizing effect produced by the exposure of
hydrophobic amino acids in the finger loop should be significant within
intact Ffh. However, proteolytic susceptibility does not necessarily
imply that the finger loop is exposed at all times. The loop could be
in a dynamic equilibrium between exposed and buried states. Further
work will be required to verify whether the loop is permanently exposed
or partially buried, for example, at the interface between the NG and M domains.
In addition to its high content of hydrophobic amino acids, the length
of the finger loop is expected to destabilize the fold of the M domain.
Statistical analyses have shown that loop regions of more than 10 amino
acids in length are rare in protein structures (16). Experimental
studies have verified that there is an inverse correlation between loop
length and the stability of proteins (17-20). Sequence analyses in
fact reveal that shortening of exposed loop regions has been used by
thermophilic organisms as an evolutionary mechanism to create more
thermally stable proteins (21). The destabilizing effect of loop length
has been rationalized theoretically by the increase in the entropic
cost of forming a loop as the length of the spacer between the ends of
the loop increases (22). It is therefore reasonable to conclude that
the retention of the finger loop in the Ffh protein through the course
of evolution reflects an essential role for this exposed hydrophobic
finger loop in the function of the SRP. This conclusion is consistent with the proposed signal sequence-binding site of Ffh (8) that implies
a role for the finger loop in the recognition of the signal peptide.
Nonetheless, more definitive experiments are needed to confirm that the
finger loop interacts with the signal sequence.
We propose that the functional necessity for the exposure of the
hydrophobic finger loop, in turn, underlies the requirement of RNA for
SRP function. The fact that RNA has been conserved through evolution in
all the cytoplasmic SRPs identified to date clearly implies an
indispensable role. Our previous studies show that RNA has the capacity
to order the tertiary structure within the M domain (11). The
proteolysis data reported here provide additional evidence that RNA
induces structural changes in Ffh. In summary, we propose that a major
role of the 4.5 S RNA is to counter the destabilizing effect of the
finger loop and thereby maintain the M domain in a functionally
competent conformation.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-helices with a long, flexible loop, denoted the "finger loop,"
between the first and second
-helices (Fig. 1A) (8). This
loop contains a number of conserved hydrophobic residues, which are
illustrated in red. A groove on the surface of the M domain
formed by the finger loop and the first, second, and fourth
-helices
has been proposed to represent the signal sequence-binding site
(8).
EXPERIMENTAL PROCEDURES
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1) was digested in the presence or absence
of ~0.7 mg ml
1 4.5 S RNA at 30 °C with
either 0.1 mg ml
1 (Fig. 2) or 5 µg
ml
1 (Figs. 4, A and B)
porcine pancreatic elastase (Roche Molecular Biochemicals) in
buffer A, 50 mM triethanolamine acetate, pH 7.5, 25 mM potassium acetate, 2.5 mM magnesium acetate,
10 mM
-mercaptoethanol, and 0.1% octaethylene
glycol monododecyl ether (Fluka). To purify the digests by Ni-NTA
chromatography, KCl and imidazole were added to them in the amounts of
300 and 5 mM, respectively, before loading onto a Ni-NTA
column; the column was washed with at least 30 column volumes of
buffer A supplemented with 20 mM imidazole and 300 mM KCl and eluted with buffer A supplemented with 500 mM imidazole and 300 mM KCl. The eluate was
concentrated by precipitation with 15% trichloroacetic acid
before analysis by SDS-PAGE. SDS-PAGE gels were stained with
Sypro-Orange dye (Molecular Probes) and analyzed on a Molecular
Dynamics PhosphorImager using ImageQuant software. To check the
consistency of the observed increase in the amount of fragment A in the
digest when RNA is present, we analyzed four pairs of
proteolysis reactions set up under identical conditions in the presence
and absence of 4.5 S RNA. To identify N-terminal protease cleavage
products by Western blotting, protease digests were electroblotted onto
nitrocellulose membranes after SDS-PAGE. After a 1-h incubation in PBS
(80 mM Na2HPO4, 20 mM NaH2PO4, and 100 mM NaCl pH 7.5)
containing 10% (w/v) nonfat dry milk and 0.05% (v/v) Tween 20, the
membranes were incubated for 2 h in the same buffer containing a
1:1000 dilution of mouse antihistidine tag antibody (Amersham Pharmacia
Biotech). After several washes in PBS containing 10% nonfat dry milk
and 0.05% Tween 20, the membrane was incubated for 2 h in the
same buffer containing a 1:1000 dilution of an anti-mouse antibody
horseradish peroxidase conjugate (Amersham Pharmacia Biotech). The
bands on the membrane were visualized using the enhanced
chemiluminescence kit (Amersham Pharmacia Biotech).
1; ellipticity measurements at 222 nm were taken at 0.1 °C intervals with a response time of 8 s
per measurement. Gel filtration analysis of the amino deletion mutant
at 200 µM protein concentration confirmed that the
protein is monomeric at concentrations more than 10 times those used
for the CD analysis (data not shown).
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Fig. 1.
The finger loop contains numerous hydrophobic
amino acids. A, structure of T. aquaticus
Ffh M domain with conserved hydrophobic residues colored red
in spacefill. The C-terminal region of the M domain that is highly
resistant to proteolytic cleavage in the presence of RNA is shown in
magenta (see Fig. 2). B, alignment of M domain
sequences from homologues of Ffh, showing the positions of the
conserved hydrophobic residues in the finger loop (in green
boxes). The numbering refers to the E. coli Ffh
sequence. The positions of helices 1-4 in the T. aquaticus
Ffh M domain and helices 1, 2a, 2b, 3, and 4 in the E. coli
Ffh M domain are shown above and below the alignment. The structure
figure was generated using the program RASTER 3D (23) with coordinates
from Protein Data Bank entry 2FFH.
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Fig. 2.
The N-terminal third of the M domain sequence
contains a highly protease-susceptible region. An SDS-PAGE of the
products of elastase digestion of the Ffh-4.5 S RNA complex reveals an
8-kDa fragment assignable to residues
Lys365-Met435 by N-terminal sequencing and
mass spectroscopy (data not shown). The protease-stable core of the M
domain, encompassing helices 2-4, is marked in magenta on
the structure in Fig. 1A.
-helical secondary
structure (Fig. 3B). As expected from our previous studies with an M domain construct purified from a proteolytic digest of Ffh
(11), thermal unfolding of the recombinant M domain construct studied
here was highly noncooperative (Fig. 3C). The near-linear decrease in ellipticity at 222 nm between 4 and 80 °C shows that the
M domain does not adopt a stable tertiary structure in solution. Despite this structural flexibility, the M domain construct used here
retained the same binding affinity for 4.5 S RNA as Ffh (Fig. 4), arguing that it is a valid
representation of the M domain within the native protein.
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Fig. 3.
The hydrophobic finger loop destabilizes the
fold of the M domain. A, schematic illustration of
truncation mutants of Ffh M domain. The amino deletion mutant (residues
Lys365-Met435) corresponds to the
magenta region on the M domain structure in Fig.
1A. This construct lacks helix 1 and the finger loop. In the
finger loop deletion mutant, the finger loop residues
Gln340 through Asp370 of the M domain have been
replaced with a GAGG linker sequence. B, CD spectra of the
amino deletion mutant, finger loop deletion mutant, and the M domain
reveal highly -helical proteins with double minima close to 208 and
222 nm. Protein concentrations are ~5 µM in all cases
in 10 mM potassium phosphate buffer at 4 °C.
C, CD at 222 nm as a function of temperature for the amino
deletion mutant (ADM), the finger loop deletion mutant
(FDM) and the M domain (MD). Thermal unfolding of
both the amino deletion mutant and the mutant lacking the finger loop
is more cooperative than the unfolding of the M domain.
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Fig. 4.
The recombinant M domain binds 4.5 S RNA with
the same affinity as Ffh. Both the M domain and Ffh form
RNA-protein complexes with slower electrophoretic mobility than 4.5 S
RNA (lanes 2 and 3 versus lane 1). The
M domain is able to compete with Ffh at comparable concentrations for
binding the RNA (lanes 4 and 5).
-helical by CD and had a significantly more
cooperative thermal unfolding profile than did the M domain (Figs. 3,
B and C). The properties of this protein were
thus entirely consistent with a destabilizing influence of the finger
loop on the M domain.
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Fig. 5.
The finger loop is surface exposed within the
context of intact Ffh. A, SDS-PAGE of products of
digestion of N-terminal dodecahistidine-tagged Ffh with a low
concentration of elastase (1 mg ml 1 Ffh, 5 µg ml
1 elastase). The crude digest is shown
in the right-hand lane, and the N-terminal
fragments separated from other fragments with Ni-NTA resin are shown in
the middle lane. For comparison purposes, a
mutant corresponding to residue 1 to 324 was constructed and loaded in
the left-hand lane. The data shown
were obtained upon proteolysis of Ffh in the absence of RNA. Comparable
fragments were generated when Ffh was digested in the presence of RNA
(data not shown). B, binding to RNA appears to enhance
protease cleavage within the finger loop. Proteolysis reactions were
incubated for 30 min under identical conditions (see "Experimental
Procedures") in the presence and absence of 4.5 S RNA. In many
repeated experiments, the N-terminal fragment A appears enhanced in
digests conducted in the presence of RNA, as shown in this
representative gel.
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ACKNOWLEDGEMENTS |
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We thank Joanna Swain for helpful discussions and critical reading of the manuscript. We are grateful to the Mass Spectrometry Facility at the University of Massachusetts, Amherst for the analysis of protein samples.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM34962.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.
¶ Present address: Howard Hughes Medical Inst., Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Univ. of Massachusetts, Rm. 814, Lederle Graduate Research Tower, 710 N. Pleasant St., Amherst, MA
01003. Tel.: 413-545-6094; Fax: 413-545-1289; E-mail:
gierasch@biochem.umass.edu.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M011130200
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ABBREVIATIONS |
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The abbreviations used are: SRP, signal recognition particle; Ni-NTA, nickel-nitrilotriacetic acid; PAGE, sodium polyacrylamide gel electrophoresis..
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