Báo cáo khoa học: An Escherichia coli twin-arginine signal peptide switches between helical and unstructured conformations depending on the hydrophobicity of the environment potx

An
Escherichia coli
twin-arginine signal peptide switches
between helical and unstructured conformations depending
on the hydrophobicity of the environment
Miguel San Miguel
1
, Rachel Marrington
2
, P. Mark Rodger
2
, Alison Rodger
2
and Colin Robinson
1,
*
1
Department of Biological Sciences and
2
Department of Chemistry, University of Warwick, Coventry, UK
The Tat system catalyzes the transport of folded globular
proteins across the bacterial plasma membrane and the
chloroplast thylakoid. It recognizes cleavable signal peptides
containing a critical twin-arginine motif but little is known of
the overall structure of these peptides. In this report, we have
analyzed the secondary structure of the SufI signal peptide,
together with those of two nonfunctional variants in which
the region around the twin-arginine, RRQFI, is replaced by
KKQFI or RRQAA. Circular dichroism studies show that
the SufI peptide exists as an unstructured peptide in aqueous
solvent with essentially no stable secondary structure. In

membrane-mimetic environments such as SDS micelles
or water/trifluoroethanol, however, the peptide adopts a
structure containing up to about 40% a-helical content.
Secondary structure predictions and molecular modelling
programs strongly suggest that the helical region begins at,
or close to, the twin-arginine motif. Studies on the thermal
stability of the helix demonstrate a sharp transition between
the unstructured and helical states, suggesting that the
peptide exists in one of two distinct states. The two non-
functional peptides exhibit almost identical spectra and
properties to the wild-type SufI peptide, indicating that it is
the arginine sidechains, and not their contribution to the
helical structure, that are critical in this class of peptide.
Keywords: signal peptide; twin-arginine translocation; Tat
system; protein transport; SufI.
The twin-arginine translocation (Tat) system operates in the
cytoplasmic membranes of most free-living bacteria and
in the thylakoid membranes of plant chloroplasts [1–3].
Operating alongside Sec-type translocases, the Tat system
functions in the transport of proteins bearing cleavable
N-terminal signal peptides (RR-signal peptides) in which a
twin-arginine motif plays a central role [4,5]. The substrate
proteins are recognized by a membrane-bound translocase
and subsequently transported, at least in some cases, in a
fully folded state [6,7]. The prime role of the Tat system
appears to be in the transport of proteins, which are either
obliged to fold prior to translocation, or which fold too
tightly for the Sec system to accommodate. Examples of
the former category include periplasmic proteins that are
exported only after binding any of a range of complex

redox cofactors, such as molybdopterin or FeS centres
[8–11]. These cofactors are inserted in the cytoplasm by
complex enzymatic processes, and it has been argued that
this must necessitate the export of these proteins in a largely,
if not fully, folded form.
Substrates bearing RR-signal peptides are recognized by
a membrane-bound translocase that consists minimally of
TatABC in most cases. Critical genes encoding these
subunits have been identified in bacteria, particularly
Escherichia coli [10–13], and in plants [14–16], and a
TatABC complex has been purified from detergent-solubi-
lized E. coli membranes [17]. The size of the purified
complex has been estimated to be in the order of 500–
600 kDa, suggesting the presence of numerous copies of
each subunit [17]. The TatBC subunits form a tight core
subcomplex in a strict 1 : 1 ratio [17] and studies on the
thylakoid system indicate that this subcomplex forms the
initial binding site for substrates, with TatA recruited at a
later stage [18,19]. Consistent with this scenario, important
residues in E. coli TatC have been identified on the
cytoplasmic side of the membrane [20,21].
Although the importance of the twin-arginine motif
has been firmly established by mutagenesis studies, RR-
signal peptides have not been characterized in structural
respects. The peptides are probably too small to form
folded globular domains but with other types of targeting
signal it has been shown that functionality is strictly
dependent on the formation of specific secondary struc-
tures. Prominent examples include Sec-type signal pep-
tides and the presequences of imported mitochondrial

proteins (e.g [22,23]). In this report, we have analyzed a
typical E. coli RR-signal peptide and we show that the
structure of the peptide differs dramatically according to
environment. Implications for the translocation process
are discussed.
Correspondence to C. Robinson, Plant Biochemistry Laboratory,
Department of Plant Biology, Royal Veterinary and Agricultural
University, 40, Thorvaldsensvej, DK-1871 Frederiksberg C,
Copenhagen, Denmark.
Fax: + 44 2476523701, Tel.: + 44 2476523557,
E-mail:
Abbreviations: MD, molecular dynamics; TFE, trifluoroethanol.
*Present address: Plant Biochemistry Laboratory, Department of
Plant Biology, Royal Veterinary and Agricultural University, 40,
Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
(Received 15 March 2003, revised 2 June 2003,
accepted 9 June 2003)
Eur. J. Biochem. 270, 3345–3352 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03710.x
Experimental procedures
Materials
Peptides, purified by HPLC, were purchased from Alta
Bioscience (Birmingham, UK).
Circular dichroism
Circular dichroism (CD) spectra are the simplest indication
of protein and peptide secondary structure. CD spectra were
collected using a Jasco J-715 spectropolarimeter equipped
with a single sample Peltier thermostatting unit. Spectra
were averaged over eight scans collected with 1 nm data
intervals, 1 nm bandwidths, 0.5 s response time and
200 nmÆs

)1
scan speed. The CD spectra as a function of
temperature were collected by monitoring at a single
wavelength (222 nm, 2 nm bandwidth) with an 8-s response
time and a ramp rate of 1 °CÆmin
)1
. At each of 20, 30, 40,
50, 60, 70, 80, and 90 °C, the temperature ramp was held
while a single wavelength scan with 4-s response time was
collected. All samples were made up to a concentration of
0.10 mgÆmL
)1
by weighing a peptide sample on a seven-
figure balance and adding the appropriate volume of
solvent. In the case of the mixed organic–aqueous solvents,
the solvent was fully mixed prior to adding to the dry
peptide. Organic solvent volumes were measured using a
syringe. Aqueous solution volumes were measured using
micropipettes. a-Helical content was estimated using a De
value of  12 mol
)1
Ædm
)3
Æcm
)1
for a 100% helical peptide
[24] and also by applying the protein CD structure-fitting
program,
CD
sstr [25]. The latter approach may only be used

as a guide as its database does not properly account for
random coil structures.
Modelling
Energy minimization calculations were performed using
QUANTA
/
CHARMM
version 28 (Accelerys Inc., Cambridge
UK) and molecular dynamics simulations with the
DL
_
POLY
package [26]. Coordinates and force field were constructed
within
QUANTA
/
CHARMM
and then exported and converted
to
DL
_
POLY
format using in-house software. Test calcula-
tions were performed on representative single configurations
using both
CHARMM
and
DL
_
POLY

to check that no
differences were observed between any of the energy
calculations. Initial configurations for the peptide were
constructed in two different ways: (a) using the
PHD
structure prediction method [27], and (b) from the lowest
energy configuration obtained from minimizing configura-
tions generated during a vacuum molecular dynamics (MD)
simulation at 500 K. The
PHD
method gives an a-helix
covering 16 amino acids, while the MD method gives a
random coil structure (see Figs 4 and 5). Initial structures
were optimized using the conjugate gradient method
(
CHARMM
). The peptide was then inserted into a solvent
box (65 A
˚
and previously equilibrated at 300 K, 1 atm),
all solvent molecules that overlapped with the peptide
removed, and three additional solvent molecules converted
to Cl
–
ions to compensate for the +3 charge of SUFI; this
resulted in 3173 water molecules, or 990 trifluoroethanol
(TFE) molecules, in a periodic truncated octahedral
simulation box. The system was then relaxed by (a)
performing a 5-ps MD simulation at 300 K, 1 atm in which
the peptide was treated as a rigid body, and then

(ii) performing a 2-ps MD simulation with a fully flexible
peptide at 2 K; these stages served to remove any strain
introduced on solvation without destroying the initial
secondary structure. A further 6 ns simulation was then
accumulated to study the response of the secondary
structure. Secondary structure was analyzed using the
STRIDE
program [28]. All MD simulations were performed
at constant temperature and pressure (NPT) using the
Nose
´
–Hoover method with thermostat and barostat relax-
ation constants of 0.5 and 1.0, respectively, and a timestep
of 2 fs. The peptide and TFE were modelled with the
CHARMM
potential, and water with the SPC model. We note
that the published TFE potentials do have some inadequa-
cies [29], and that the
CHARMM
potential can underestimate
the stability of a-helices relative to other force fields [30], but
overall the model is reasonable. Long range forces were
truncated at 10 A
˚
, and the reaction field method used to
correct for long-range electrostatic effects.
Secondary structure prediction
Secondary structure was predicted using the
PSIPRED
[31],

JPRED
[32],
PROF
[33] and
PHD
[27] secondary structure
prediction programs.
Results
Structures of wild-type and nonfunctional SufI signal
peptides
The overall aim in this study was to analyze the structural
characteristics of a wild-type RR-signal peptide in both
aqueous solvent and membrane-mimicking environments,
as well as in some intermediate environments. The
objective was to identify the secondary structure charac-
teristics immediately after synthesis (in aqueous medium,
as the targeting process is post-translational at least in the
vast majority of cases) and once bound either to the
membrane or the translocase (see below). A possibility
addressed in this study was that the twin-arginine motif
somehow may contribute significantly to the secondary
structure, and we therefore also analyzed mutant variants
that are not recognized by the Tat system. In this way, we
sought to determine whether the significance of the twin-
arginine motif stems from the nature of the sidechains and/or
its ability to promote a given form of secondary structure.
We chose SufI as an example of a typical E. coli Tat
substrate. SufI is synthesized with an apparently typical
RR-signal peptide with the sequence shown in Fig. 1. Two
variants are also shown; in one (SufI-KK), the twin-arginine

motif is replaced by twin-lysine, which causes either a
complete or near-complete block in translocation by the Tat
pathway in both chloroplasts and bacteria [4,5,34]. In the
other, the region around twin-arginine motif, RRQFI, is
replaced by RRQAA (SufI-AA). While the effects of this
double mutation have not been tested in bacterial systems,
studies on the thylakoid system have demonstrated the
critical importance of a highly hydrophobic residue at the
second or third positions after the twin-arginine motif [35].
3346 M. San Miguel et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Bacterial RR-signal peptides invariably contain such a
residue, typically phenylalanine, leucine, isoleucine or
valine, at one or both of these positions [8] and the SufI-
AA peptide shown in Fig. 1 is therefore strongly predicted
to be nonfunctional. The SufI peptide has been synthesized
chemically and shown to competitively inhibit the Tat-
dependent transport of proteins into inverted E. coli vesicles
[36], and to interact with Tat complexes in detergent [37].
This indicates that the peptide is functional in isolation and
we used the same peptide in the present study in order to
examine the structure of the signal at various stages. The
KK mutant peptide does not affect binding of wild-type
substrate suggesting that the RR motif is required for
substrate binding.
The SufI RR-signal peptide is unstructured in aqueous
solvent but a-helical in membrane-mimetic environments
The secondary structure of the SufI RR signal and the
mutant variants was analyzed by CD. Figure 2 shows their
structures in aqueous solvent (pH ¼ 7) and two membrane-
mimetic environments, namely SDS micelles and water/

TFE systems. A range of alcohols in which the peptide was
soluble were also investigated and spectra in methanol and
ethanol shown for SufI. The data obtained with the wild-
type peptide in water (Fig. 2A) show a spectrum character-
istic of an unstructured peptide with a negative maximum
below 200 nm and no signal at 220 nm. The spectra in the
other solvent systems are typical of a-helices, though of
Fig. 1. Structure of the SufI signal peptide.
Shown are the amino acid sequences of the
E. coli SufI twin-arginine signal peptide,
together with the sequences of two mutant ver-
sions used in this study. The essential
twin-arginine motif is shown as bold italics.
Fig. 2. CD spectra of SufI signal peptides in
aqueous and apolar solvents. (A) CD spectra
were taken using the wild-type SufI signal
peptide in solvents as indicated in the figure.
Further details are given in Experimental
procedures. (B) Comparison of the wild-type
peptide with the SufI-KK and SufI-AA pep-
tides (denoted as ÔKKÕ or ÔAAÕ, respectively, in
inset to graph). CD spectra were taken in
water, water/TFE (50 : 50 v/v) and TFE.
Ó FEBS 2003 Structural characteristics of Tat-specific targeting signals (Eur. J. Biochem. 270) 3347
magnitude significantly less than expected for 100% helix
(De  12 mol
)1
Ædm
)3
Æcm

)1
at 208 nm and 222 nm) even in
TFE where the helical content is  45%. The alcohols
ethanol, propan-1-ol and butan-1-ol gave the same spec-
trum within experimental error down to 195 nm, with an
a-helix content of  25%. Methanol induces a noticeably
more helical content ( 40%), similar in magnitude to a
50% TFE/water solvent. A 0.5% w/v SDS solution resulted
in a structure similar to that observed with ethanol.
Figure 2B shows the spectra obtained with the SufI-KK
and SufI-AA peptides. In both cases, the spectra are almost
identical to those of the wild-type indicating that the twin-
arginine motif does not contribute specific a-helical prop-
erties in this context. Compared with SufI, the 208 nm
region of the spectra indicates a slight increase in helical
character for SufI-AA and a slight decrease for SufI-KK in
TFE, whereas at 222 nm in water/TFE SufI-AA has less
intensity and SufI-KK is the same as SufI. This suggests that
the mutant helices are perhaps of slightly different form.
The SufI-AA random coil spectrum (water) also differs
slightly from the others.
Thermal stability of the a-helix: evidence for two
distinct states
The CD spectra of SufI collected in 50 : 50 water/TFE as
a function of temperature show a well-defined nonzero
isosbestic point at 201 nm (Fig. 3A). This indicates that the
system is in one of two states, presumably a-helical and
random coil. The fact that there is no sharp transition
during the temperature ramp (Fig. 3B) suggests that we
have a temperature dependent equilibrium between the two

states rather than any sort of concerted transition. The
situation with the two mutants is very similar, though there
is only an approximate isosbestic point in each case, with
KK being slightly the worse (data not shown). Although
there are not enough data to enable us to determine the
cause of this, the most likely cause is a slight variation in
helical forms present in the solution.
Location of the helical segment(s) in hydrophobic milieu
The SufI RR-peptide clearly contains substantial amounts
of helical structure in apolar environments and, because the
CD data do not indicate the location of this structure within
the 27-residue peptide, we used additional methods in order
to pinpoint the likely location(s). First, the
PSIPRED
,
JPRED
,
PROF AND PHD
secondary structure prediction programs
were used, and typical predictions are shown in Fig. 4A. All
four programs predict substantial a-helical content (26, 48,
56 and 59%, respectively) and it is notable that these regions
encompass the twin-arginine motif in each case, with the
RR motif usually positioned at or near the beginning of the
helical section.
As a complementary technique we carried out a detailed
molecular modelling simulation of SufI RR in both water
and TFE. Simulations were performed using two different
initial peptide conformations, one taken from the
PHD

structure prediction (59% a-helix; diagram 1 of Fig. 4B)
and one with essentially a random coil secondary structure
(the lowest energy structure from the conformational
search; not shown). The simulated secondary structure
from these two initial configurations was found to have
converged after about 1 ns, indicating that the subsequent
structures were stable and not merely an artefact of the
simulation timescale, nor of the starting geometry.
The results of the simulations are in semiquantitative
agreement with the CD experiments. No a-helix was found
to persist in water, and indeed the 16 amino acid helix in
the
PHD
initial structure completely disappeared within
 200 ps. Typical peptide conformations from the end of a
simulation in water are shown in diagrams 3 and 4 of
Fig. 4B. In contrast, the long time behaviour of the
simulations in TFE showed about 20% a-helix (not shown).
This value is lower than the CD experiments suggest
( 45%). Given the reservations about the TFE potential
discussed earlier, this probably indicates that the TFE
potential is slightly too hydrophilic in character so that only
the most significant a-helix-forming tendency emerges in the
simulation. While some fluctuation in the length of the helix
was observed, the RR motif was consistently found at the
beginning of the helix.
Discussion
Numerous RR-signal peptides have been characterized in
terms of primary sequence and it is now clear that, in most
cases, they conform to a standard basic model in which

three distinct domains can be identified: a charged
N-terminal (N-) domain, hydrophobic core (H-) domain
and more polar C-terminal domain terminating with a
consensus motif that specifies cleavage by the processing
peptidase. Key structural determinants are located at the
boundary between the N-domain and the hydrophobic
core. In plants, RR-signal peptides contain an essential
twin-arginine motif plus a highly hydrophobic residue two
or three residues towards the C-terminus. Typically, the
sequence RRXXL/F/I/M or RRXL/F/I/M is found [35]. In
E. coli, a slightly different consensus is observed, namely
RRxFLK [8]. However, E. coli RR-signal peptides are
efficiently recognized by plant thyalkoids indicating a highly
conserved interaction between the signal peptide and the
Tat translocation apparatus [38,39].
A surprising point is that Tat-dependent signal peptides
closely resemble Sec-type signals in overall terms. Sec signals
likewise contain the three domains described above and
the only immediately notable difference is that the basic
residue(s) at the N–H domain junction can be either
arginine or lysine. In bacteria, Sec signals also tend to be
more hydrophobic than RR-signal peptides. Nevertheless,
these similarities raise the possibility that the signals may not
always direct targeting by the correct translocation path-
way, at least initially.
While the primary sequences of Tat-type signal peptides
are now relatively well established, very little is known of
their secondary structures. This is an important issue for
two reasons. Firstly, it is now clear that the twin-arginine
motif plays a crucial role in the translocation process and

this could be either (a) because its sidechains are
specifically recognized by the Tat translocase, and/or (b)
because it confers a specific secondary structure determin-
ant that is important in the context of the remainder of
the peptide. Second, the targeting of Tat substrates is in
most (if not all) cases an obligatorily post-translational
3348 M. San Miguel et al. (Eur. J. Biochem. 270) Ó FEBS 2003
process, raising the possibility that the signal may adopt
different structures before and after reaching the target
membrane.
In this study, we have used the SufI signal peptide as a
model and the data show that this signal can adopt two
radically different structures. In aqueous solution, our data
show that the signal contains essentially no stable secondary
structure. CD is particularly sensitive for the detection of
a-helices, and the data show quite clearly that the signal has
no stable helical elements, presumably because of compe-
tition for hydrogen bonding by water molecules. After
release from the ribosome, Tat signal peptides are therefore
likely to exist as unstructured peptides. In more hydro-
phobic environments, namely 50 : 50 water/TFE or SDS
micelles, the structure is very different and the wild-type
SufI peptide contains approximately 40 and 25% a-helical
structure, respectively. Both secondary structure predictions
and MD simulations of SufI in TFE (explicit solvent)
strongly suggest that the helical structure is located in the
centre section of the peptide, starting either just before or at
Fig. 3. Thermal stability of the helical regions
in SufI signal peptides. (A) The wild-type SufI
peptide in TFE/water (50 : 50 v/v) was ana-

lyzed by CD and spectra were taken at the
temperatures indicated. (B) Similar CD spec-
tra were recorded using the SufI-AA and SufI-
KK peptides (not shown) and the graph shows
plots of the change in De at 201 nm as a
function of temperature for all three peptides.
Ó FEBS 2003 Structural characteristics of Tat-specific targeting signals (Eur. J. Biochem. 270) 3349
the twin-arginine motif. The MD simulations also indicate
that the formation of an a-helix is very rapid in a
hydrophobic environment.
While the twin-arginine is strongly predicted to lie at or
near the starting point for the helical domain, our data
indicate that it does not itself dictate the formation or extent
of the helical sequence in a specific manner because a
mutant version of the peptide containing twin-lysine
contains almost exactly the same amount of a-helical
structure. This finding strongly suggests that the real
significance of the twin-arginine motif is concerned with
protein–protein interactions involving the two extensive
arginyl sidechains.
On the basis of these data we suggest the following model
for the structures of Tat signal peptides during the overall
translocation process (Fig. 5). Immediately after synthesis,
the signal peptide probably exists in equilibrium between
unstructured peptide and a more structured peptide con-
taining substantial a-helix in the core region. While in
aqueous solvent, the unstructured peptide predominates, to
the point where the helical form is essentially undetectable.
There is no evidence for the involvement of any soluble
Fig. 4. Predicted secondary structure in the SufI signal peptide. (A) Secondary structure for the wild-type SufI peptide (WT) predicted using the

PSIPRED
,
JPRED
,
PROF
and
PHD
programs. C indicates random coil, H an a-helix and E an extended or sheet motif. The percentage helical content of
the peptides is shown on the right. (B) 3D representations (ribbons mode on the left and bonds mode on the right) of the initial structures for the WT
peptide obtained from the
PHD
program (1 and 2) and final configurations from an MD simulation in water (3 and 4); the twin-arginine motif has
been depicted using a space-filling representation.
3350 M. San Miguel et al. (Eur. J. Biochem. 270) Ó FEBS 2003
chaperone-type proteins and we therefore believe that the
next protein–protein interaction is with the Tat translocase,
probably by one of two pathways as shown in Fig. 5. In
pathway A, the helical structure is promoted by interaction
with the target membrane. There is now good evidence that
a-helix formation is strongly promoted within the mem-
brane interfacial region [40] and the precursor protein may
then migrate laterally on the membrane surface until it
contacts the translocase. In pathway B, the signal interacts
directly with the translocase and the helical conformation is
generated either during entry into the interfacial region or
after the initial interaction with the binding site. Any form
of typical binding site/groove probably also favours the
formation of secondary structure, and the enormously
specific signal peptide–translocase interaction would be
difficult to achieve if the peptide were largely unstructured.

We therefore contend that the signal peptide must be in the
helical form when docked onto the translocase binding site.
A precedent is the interaction of mitochondrial targeting
peptides with the Tom20 receptor, where the targeting
signal is likewise unstructured in aqueous solution but in the
form of an amphipathic a-helix when bound to the receptor
[41,42]. Further work should determine whether the same
applies to the Tat-dependent translocation process.
Acknowledgements
This work was supported by Engineering and Physical Sciences
Research Council grant GR/R36503 to CR and PMR.
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Fig. 5. Models for the structures of signal peptides during the overall
Tat-dependent translocation process. Immediately after synthesis, the
signal peptide is exposed to an aqueous environment where it is in
equilibrium between the unstructured (1) or substantially a-helical (2)
conformations. The equilibrium is strongly in favour of the unstruc-
tured peptide, probably because of competition for H-bonding by
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membrane and a-helix formation is promoted within the inter-
facial region, after which the precursor protein migrates laterally until

contact with the translocase is made. In B, the precursor protein
interacts directly with the translocase, in which a-helix formation may
be promoted within the binding site.
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