doi:10.1016/S0022-2836(02)00940-3 available online at http://www.idealibrary.com on
w
B
J. Mol. Biol. (2002) 323, 463–473
TROSY-NMR Studies of the 91 kDa TRAP Protein
Reveal Allosteric Control of a Gene Regulatory Protein
by Ligand-altered Flexibility
Craig McElroy1, Amanda Manfredo2, Alice Wendt2, Paul Gollnick2 and
Mark Foster1,3*
1
Ohio State Biochemistry
Program, Department of
Biochemistry, The Ohio State
University, Columbus, OH
43210, USA
2
Department of Biological
Sciences, State University of
New York, Buffalo, NY 14260
USA
3
Biophysics Program and
Protein Research Group
Department of Biochemistry
The Ohio State University
Columbus, OH 43210, USA
The tryptophan biosynthesis genes of several Bacilli are controlled
through terminator/anti-terminator transcriptional attenuation. This process is regulated by tryptophan-dependent binding of the trp RNA-binding attenuation protein (TRAP) to the leader region of the trp operon
mRNA, precluding formation of the antiterminator RNA hairpin, and
allowing formation of the less stable terminator hairpin. Crystal structures
are available of TRAP in complex with tryptophan and in ternary complex
with tryptophan and RNA. However, no structure of TRAP in the absence
of tryptophan is available; thus, the mechanism of allostery remains
unclear. We have used transverse relaxation optimized spectroscopy
(TROSY)-based NMR experiments to study the mechanism of ligandmediated allosteric regulation in the 90.6 kDa 11-mer TRAP. By recording
a series of two-dimensional 15N-edited TROSY NMR spectra of TRAP
from the thermophile Bacillus stearothermophilus over an extended range
of temperatures, we have found tryptophan binding to be temperaturedependent, in agreement with the previously observed temperaturedependent RNA binding. Triple-resonance TROSY-based NMR spectra
recorded at 55 8C have allowed us to obtain backbone resonance assignments for uniformly 2H,13C,15N-labeled TRAP in the inactive form and in
the active form (free and bound to tryptophan). On the basis of liganddependent differential line-broadening and chemical shift perturbations,
coupled with the results of proteolytic sensitivity measurements, we
infer that tryptophan-modulated protein flexibility (dynamics) plays a
central role in TRAP function by altering its RNA-binding affinity.
Furthermore, because the crystal structures show that the ligand is buried
completely in the bound state, we speculate that such dynamic behavior
may be important to enable rapid response to changes in intracellular
tryptophan levels. Thus, we propose that allosteric control of TRAP is
accomplished by ligand-altered protein dynamics.
q 2002 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: TRAP; TROSY; dynamics; allostery; tryptophan
Introduction
Transcriptional control of the expression of
many genes in bacteria is mediated by attenuation,
involving competition between mutually exclusive,
overlapping terminator and anti-terminator RNA
secondary structures in the 50 leader region of the
Abbreviations used: TRAP, trp RNA-binding
attenuation protein; TROSY, transverse relaxation
optimized spectroscopy.
E-mail address of the corresponding author:
[email protected]
nascent mRNA. In several Bacilli, attenuation
control of the trp operon is regulated by the trp
RNA-binding attenuation protein (TRAP), which,
by binding to the nascent mRNA, controls which
hairpin forms.1 – 5 In the absence of tryptophan,
TRAP does not bind to its target RNA and a more
stable anti-terminator structure forms, allowing
transcriptional read-through of the entire operon
(Figure 1). Upon binding tryptophan, TRAP
becomes activated to bind to the conserved 11
G/UAG triplet repeats in the leader region of the
trp operon.6 Because its binding site partially overlaps the anti-terminator sequence, TRAP binding
0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved
464
TROSY-NMR of Free and Tryptophan-bound TRAP
Figure 1. The transcriptional attenuation mechanism of TRAP. The 50 leader region preceding the trp operon is
shown numbered according to the transcription start site, with the triplet repeats of the TRAP binding site circled.
Upon transcription initiation, in the absence of tryptophan, the anti-terminator hairpin structure forms (indicated by
arrows A and B), and transcription proceeds un-attenuated. In the presence of tryptophan, TRAP becomes activated
to bind its cognate sequence, which overlaps the anti-terminator hairpin. In so doing, it favors formation of the terminator structure (arrows C and D), resulting in attenuation. The terminator and anti-terminator hairpins are mutually
exclusive, as the boxed ACCC sequence forms part of both stems. Although the crystal structure of activated (holo-)
TRAP is known, the structure of inactive (apo-)TRAP is not available, and thus the mechanism of allosteric regulation
has been unclear.
to RNA prevents the formation of the anti-terminator structure and thus favors formation of
the terminator structure, prohibiting further
transcription.1
The crystal structures of TRAP from B. subtilis
and from B. stearothermophilus in binary complex with L -tryptophan and of TRAP from
B. stearothermophilus in ternary complex with
5,7,8
L -tryptophan and RNA have been reported.
These structures reveal 11 identical protein subunits arranged in a donut-like fashion, with each
monomer related by an 11-fold rotational axis of
symmetry (Figure 2(a)). The oligomer is comprised
of 11 anti-parallel b-sheets consisting of seven
strands each. The 11 tryptophan ligands are buried
completely in a hydrophobic pocket at the interface between subunits, and constitute , 14% of the
inter-subunit contact surface (Figure 2(b)). The
crystal structure of TRAP in complex with RNA5,8
reveals that the latter wraps entirely around
the outside of the protein, making specific base/
residue interactions with the GAG repeats of the
RNA via conserved Phe32, Glu36, Lys37, Lys56
and Arg58 residues of each subunit in the protein;
with the exception of a few side-chains in the
protein– RNA interface, the protein structure is
essentially unchanged by binding the nucleic
acid. Although these structures reveal the intermolecular interactions that enable specific tryptophan and RNA recognition, they do not reveal a
mechanism by which the allosteric ligand regulates
RNA binding by TRAP, and to date no crystallographic data are available on TRAP in the absence
of tryptophan.
The transition of TRAP between the inactive and
active states involves ligand binding/dissociation,
but no obvious path to solvent can be inferred
from the available crystal structures (see Figure
2(b)). In this context, three potential regulatory
mechanisms can be envisioned to transmit the
allosteric change from the ligand-binding site
to the RNA-binding surface: I, ligand-dependent assembly/disassembly of the oligomer; II, a
ligand-induced switch between distinct conformational states; or III, a ligand-dependent change
in protein dynamics and/or stability. The key
requirements of the correct mechanism are that it
should allow for the ligand to enter and to exit its
binding site, and simultaneously induce a change
(structural and/or dynamic) in the RNA-binding
surface of the protein.
Ligand-induced changes in oligomeric state have
been proposed to regulate other transcriptional
regulators, including the Escherichia coli maltose
regulon activator MalT,9,10 the Agrobacterium tumefaciens quorum-sensing transcriptional regulator
TraR,11,12 and heterodimeric retinoic acid receptors
RAR and RXR.13 The assembly mechanism for
TRAP is ruled out by size-exclusion and sedimentation measurements14 (and the data reported
465
TROSY-NMR of Free and Tryptophan-bound TRAP
between subunits, two possible conformational
transitions could be envisioned as regulating
sequence-specific RNA binding: a reorientation
of the subunits (IIa) or a local conformational
change (IIb). Although at times at odds with the
conclusions of crystallographic analyses, a few
experimental examples have argued for a role for
ligand-induced changes in protein flexibility in
mediating control of gene regulatory proteins
(mechanism III), including the lac19 and trp,20 – 22
repressors and RXR.23
We have sought to characterize the mechanism
of allosteric regulation of the B. stearothermophilus
TRAP protein. Using transverse relaxation
optimized spectroscopy (TROSY)-based NMR
methods,24 – 29 we have obtained backbone resonance assignments for TRAP in the presence and
in the absence of the regulatory ligand, tryptophan.
From ligand-dependent differential line-broadening and chemical shift perturbations, supported
by proteolytic-sensitivity measurements, we have
found that TRAP exhibits different dynamic
behavior in the inactive (apo) and active (tryptophan-bound/holo) states. We therefore propose
that ligand-mediated modulation of protein
dynamics controls TRAP function and thereby
gene expression.
Results
Quality of spectra
Figure 2. (a) Ribbon diagram of the tertiary and
quaternary structure of the undecameric TRAP in ternary complex with L -tryptophan and RNA.8 Each subunit
is displayed in a different color with the tryptophan
residue shown in green and RNA in yellow. (b) The
allosteric tryptophan ligand is buried in the interface
between two subunits in the ternary complex of TRAP
with tryptophan and RNA (1CS9). As discussed in
the text, NMR data indicate that protein dynamics
are responsible for providing access to solvent and
mediating RNA binding. The RNA is yellow, the tryptophan residue is green, and the backbone of residues
undergoing chemical exchange dynamics in the TRAP–
tryptophan complex is red. The arrow indicates the site
of cleavage by trypsin in apo-TRAP.
here), which indicate that the oligomeric state of
TRAP remains unchanged in the absence of
tryptophan.
Ligand-induced conformational switches leading to protein activation for DNA binding (mechanism II) have been proposed for transcriptional
regulators including CAP,15 the trp repressor16 and
the nuclear hormone receptor family.17,18 In TRAP,
considering that the ligand binds in the interface
Although TRAP is too large for analysis by conventional NMR methods (90.6 kDa), through the
use of TROSY-based pulse sequences, uniform
deuteration, and elevated temperatures afforded
by the thermophilic B. stearothermophilus protein,
the spectral quality for both holo and apo-TRAP is
quite reasonable (Figure 3). NMR spectra recorded
at 55 8C are in agreement with the 11-fold symmetry observed in the crystal structure, exhibiting
only the spectral complexity expected for the
monomer, although the TROSY line-widths are
somewhat broader than might be expected
(Figures 3 and 5(a) and (b)).24 Resonance linewidths of apo-TRAP are generally comparable to
those in holo-TRAP, supporting the conclusion
that the oligomer does not disassemble in the
absence of the ligand (mechanism I).9
Notably, a selection of resonances observed in
spectra recorded in the presence of tryptophan is
missing from spectra recorded in the absence of
the ligand (labeled with an asterisk in Figure 3).
We interpret the disappearance of these signals as
resulting from extreme conformational exchange
broadening: that is, a portion of the protein is
exchanging between two or more conformations
with different chemical shifts, on an intermediate
time-scale (milli- to microsecond) relative to
the difference in the resonance frequencies of the
individual states. The implications of this observation are discussed below.
466
TROSY-NMR of Free and Tryptophan-bound TRAP
Figure 3. Overlay of the 2D 1H,15N TROSY spectra of TRAP recorded in the presence (black contours) and in the
absence (red contours) of tryptophan. Corresponding backbone amide assignments are displayed next to the peaks.
Resonances from holo-TRAP that are broadened beyond detection in the triple resonance TROSY spectra of apoTRAP are indicated with an asterisk.
Resonance assignments
25 – 29
TROSY-based triple resonance spectra
recorded on uniformly, triply labeled (2H,13C,15N)
TRAP allowed for assignment of 77% of the backbone resonances in the holo-TRAP, (C.M., A.M.,
A.W., P.G. & M.P.F., unpublished results) but
the above-mentioned conformational exchange
line-broadening (particularly near the tryptophan-binding site) resulted in only 49% of the
backbone resonances being assigned in apoTRAP. Additional unassigned residues in apo
and holo-TRAP correspond to regions in the
protein for which there is no electron density
in the crystal structure or that exhibit the
largest B-values.5 Backbone amide assignments
of holo and apo-TRAP are as indicated in
Figure 3.
Tryptophan binding is temperature-dependent
Filter-binding assays have shown that RNAbinding in B. stearothermophilus TRAP is temperature-dependent, with optimal affinity at 70 8C.5
The 15N-edited heteronuclear single quantum
coherence (HSQC) and TROSY spectra recorded at
this temperature on recombinantly expressed and
purified TRAP exhibit a single cross-peak in the
indole H11 region that corresponds to tryptophan
that co-purifies with the protein†. In contrast, in
spectra recorded at 25 8C (Figure 4, first panel),
we observe a line-shape, intensity, and chemical
shift for the indole H11 cross-peak that matches
that of free tryptophan;30 we infer that at this
temperature the tryptophan is largely free in solution and tumbling much faster than the protein.
As the temperature is increased to 55 8C, the signal
from free tryptophan is replaced by a downfieldshifted resonance with a line-width matching that
of the other signals in the spectrum. This resonance
thus serves as an indicator of the binding state of
the ligand and establishes that tryptophan binding
is temperature-dependent. Although the temperature at which no signal for the free tryptophan can
† Assignment of the tryptophan H11 resonance was
confirmed by addition of an excess of unlabeled
tryptophan to the sample. The unlabeled tryptophan
displaced the bound labeled tryptophan, resulting in a
shift of the resonance to that expected for free
tryptophan (Figure 4, last panel)30; no other change in the
spectrum was observed.
467
TROSY-NMR of Free and Tryptophan-bound TRAP
Figure 4. Expanded region of
2D 1H,15N HSQC spectra of TRAP
recorded in the presence of tryptophan, in 5 deg. C intervals from
25 to 75 8C. The data show that
tryptophan binding is temperaturedependent, being bound completely at 55 8C but largely unbound
at 25 8C. The asymmetry of the
signal from bound tryptophan at
intermediate temperatures reflects
the partial occupancy of the ligandbinding sites, consistent with the
absence of positive cooperativity.
To confirm that the intense signal
in the 25 8C spectrum corresponds
to the free amino acid, a spectrum
was recorded at 55 8C after the
15
N-labeled tryptophan was displaced from its binding site in
TRAP by the addition of excess
unlabeled tryptophan (last panel).
Figure 5. The top panel corresponds to the secondary structure
of TRAP with the b-sheets A– G
labeled. (a) and (b) Proton linewidths (Hz) measured from the 2D
1
H,15N TROSY spectra of TRAP
recorded (a) in the presence and
(b) in the absence of tryptophan.
(c) Weighted average chemical shift
perturbations, Dav(HNCa), induced
by the addition of tryptophan.
(d) and (e) Shortest approach distance (Å) between TRAP and
(d) bound tryptophan or (e) RNA
in the crystal structure of the
ternary complex (1CS9).8
be detected is approximately 55 8C (Figure 4), as
the temperature is increased further to the
maximal RNA-binding temperature (70 8C),5
additional chemical shift changes are observed.
The coincidence of the temperature of
maximal RNA binding with the endpoint of
changes in the tryptophan H11 resonance is
consistent with the central role of the allosteric
ligand in regulating RNA binding. Furthermore,
the asymmetric peak shape of the indole
resonance
at
intermediate
temperatures
is consistent with the presence of a mixture of
TRAP molecules with incompletely occupied binding sites, in agreement with the experimentally
determined lack of cooperativity in tryptophan
binding for this protein.5
Tryptophan binding alters conformational
exchange dynamics in TRAP
Examination of the resonance line-widths in
spectra recorded with and without tryptophan
(Figures 3, 5, and 6(a) and (b)) provided valuable
insights into the ligand-mediated changes in
the protein dynamics. Because they are affected
strongly by chemical exchange, resonance linewidths often yield qualitative insights into regions
of the protein that are dynamic on the micro- to
millisecond time-scale. Comparison of the amide
1 N
H resonance line-widths in apo and holo-TRAP
revealed a strong correlation between localized
changes in flexibility and distance to the tryptophan-binding site (Figures 5 and 6).
468
TROSY-NMR of Free and Tryptophan-bound TRAP
Figure 6. Tryptophan-mediated
spectral perturbations mapped
onto a “worm” diagram of the crystal structure of B. stearothermophilus
TRAP in ternary complex with
tryptophan and RNA (1CS9).8
Neighboring subunits in the undecamer are cyan, the tryptophan
ligand is green, and RNA is yellow.
Residues whose resonances are
severely broadened by conformational exchange are red. (a) and
(b) Proton line-widths measured
from 2D 15N-edited TROSY spectra
of TRAP (a) in the absence and (b)
in the presence of tryptophan,
mapped onto the backbone with
the radius of the worm rendered in
proportion to the line-widths. (c)
Chemical
shift
perturbations
induced by tryptophan mapped
onto the backbone by a linear color
ramp corresponding to the magnitude of the observed weighted average chemical shift differences,
Dav(HNCa), for the backbone amide
proton, nitrogen and Ca resonances
(white, no change; blue, maximal
change). (a) In the absence of
tryptophan, most of the resonances
from the ligand-binding site are
severely exchange-broadened (red),
as is much of the RNA-binding surface formed by strands bC and bE.
(b) Resonances from both of these
strands can be assigned in activated
TRAP. (c) In addition to liganddependent line-broadening, the largest (measurable) ligand-induced
chemical shift perturbations map to
strand bC, reflecting transduction
of the signal from the ligand-binding site to the RNA-binding surface.
Not surprisingly, the largest tryptophan-dependent changes in line-width occur for residues near
its binding site in TRAP. For instance, the crosspeaks corresponding to residues from the end of
strand bB through most of strand bC (Gly23Lys37‡) and in the loop between strands bD and
bE through most of strand bE (Gln47-Lys56) are
the broadest signals in the holo-TRAP spectrum,
‡ Residue numbers used here correspond to those of
B. subtilis TRAP, matching the scheme used in the crystal
structure B. stearothermophilus TRAP (1C9S.PDB).
and are broadened beyond detection in the apoTRAP spectrum. In addition, line-widths for residues in strand bF (Lys60-His67), which forms the
Figure 7. Effect of tryptophan on TRAP sensitivity to
trypsin. Cleavage was monitored by tris-tricine SDS-10 –
20% PAGE after digesting apo- (lanes 1 – 7) and holoTRAP (lanes 8 – 14) for 0, 1, 2.5, 5, 15, 30 and 60 minutes
with 5% (w/w) trypsin.
469
TROSY-NMR of Free and Tryptophan-bound TRAP
shift changes (Figures 3 and 5). Mapping the
magnitude of the chemical shift perturbations
onto the crystal structure of the ternary TRAP
complex8 (Figure 6(c)), shows that residues constituting the core of the protein complex are largely
unperturbed. On the other hand, the largest
measurable chemical shift perturbations map to
Glu38 and Asp39 on strand bC, two residues that
are far removed from the tryptophan-binding
site (Figures 5 and 6), but constitute part of the
RNA-binding surface.8
Discussion
Figure 8. A putative model for the role of protein
dynamics in the TRAP allosteric regulation cycle. (a) In
the inactive state of TRAP (apo) the RNA-binding surface is dynamically disordered and lacks the appropriate
architecture for nucleic acid binding. (b) Tryptophan
binding activates TRAP for RNA binding by inducing
local protein folding and stabilizing the RNA-binding
scaffold. (c) The ternary complex of TRAP with tryptophan and RNA. (d) Dynamic exchange of tryptophan
enables destabilization of the TRAP – RNA interaction
and loss of transcriptional attenuation. In (b) and (c),
protein dynamics are damped but still present and
necessary, enabling ligand exit and response to intracellular ligand concentration.
base of the donut structure and is far removed
from the tryptophan-binding site, are narrower in
the holo protein, consistent with an overall ligandinduced rigidification of the protein oligomer
(Figures 5, and 6(a) and (b)).
Further evidence for local conformational disorder in TRAP in the absence of tryptophan was
obtained by assaying the susceptibility of apo and
holo-TRAP to digestion with trypsin. Indeed, apoTRAP was cleaved readily by trypsin, whereas
holo-TRAP was highly resistant to cleavage
(Figure 7). Reversed-phase HPLC purification of a
trypsin-generated peptide fragment followed by
electrospray mass spectrometry confirmed that
cleavage takes place after Arg31 (5105 Da
observed, 5104.8 Da expected for Phe32-Lys76),
which is located in the loop between strands bB
and bC (Arg26-Phe32) that comprises part of
the tryptophan-binding site (Figures 2(b), and 6(a)
and (b)).
Chemical shift mapping
Comparison of the spectra of TRAP recorded
with and without tryptophan reveals differential
line-broadening, and many localized chemical
We have used TROSY-based NMR methods to
study the active (tryptophan-bound/holo) and
inactive (apo) states of the 90.6 kDa undecameric
TRAP complex with the objective of revealing
the mechanism of ligand-induced activation (i.e.
allostery). In spite of the large size of this oligomeric protein, its thermal stability and 11-fold symmetry enabled us to obtain backbone resonance
assignments (albeit incomplete) in both the active
state and the inactive state. From analysis of
ligand-dependent chemical shift changes and
differential line-broadening, we propose that
ligand-modulated changes in protein dynamics
play a central role in regulating the function of
TRAP.
Protein loop motion plays a pivotal role in
allosteric regulation
Although further work will shed light on the
details of the relevant thermodynamics and
dynamics, the observed ligand-dependent localized line broadening (Figures 3(b), and 5(a) and
(b)), together with increased proteolytic sensitivity
(Figure 7), argue strongly that apo-TRAP is more
flexible than holo-TRAP; this is consistent with
the failure to date of efforts to obtain suitably
diffracting crystals of apo-TRAP (P.G. & A. Antson,
unpublished results). Analysis of the NMR data
for holo and apo-TRAP mapped onto the crystal
structure of the ternary TRAP –Trp-RNA complex
(Figure 6(a) and (b)),8 allows us to conclude that
flexibility in the ligand-binding site (largely the
bB-bC and bD –bE loops) is propagated to the
RNA-binding surface in apo-TRAP.
A principal objective of these studies was to
distinguish between the possible mechanisms for
tryptophan-mediated allosteric regulation of RNA
binding by TRAP. The absence of large chemical
shift changes in the protein– protein interfaces
(Figures 5(c) and 6(c)) argues against ligandinduced changes in subunit orientation, while the
observed localized line-broadening and increased
proteolytic sensitivity are consistent with liganddependent changes in flexibility (in support of
mechanism III). Furthermore, many of the
residues experiencing altered dynamics or shift
perturbations were previously identified as being
470
important for RNA binding. For instance, Glu36,
Lys37, and Lys56 are among the dynamically disordered (i.e. exchanged-broadened) residues in
apo-TRAP (Figures 5(b) and (c), and 6(a) and (c)),
and were all shown to interact specifically with
the RNA in the crystal structure,8 while Lys37 and
Lys56 were shown to be critical for RNA binding
in alanine scanning mutagenesis studies.31 The
strong correlation between the NMR data and the
crystallographic and biochemical data has enabled
us to develop a model for the allosteric behavior
of the protein.
A model for allosteric control of TRAP by
ligand-altered protein dynamics
In the context of the available crystallographic
and biochemical data, the evidence presented here
for ligand-dependent localized flexibility allows
us to propose a model (Figure 8) in which tryptophan modulation of protein dynamics plays a
central role in TRAP function by altering RNAbinding affinity and by enabling rapid response to
intracellular tryptophan levels. In the inactive
form of TRAP (populated at low concentrations of
tryptophan), the oligomeric structure is well
formed,14 but the RNA-binding surface is conformationally dynamic/disordered (Figure 8(a)). In
this state, the protein cannot bind RNA, the antiterminator hairpin is retained, and transcription of
the trp operon proceeds un-attenuated. In addition
to serving as a mechanism to impede RNA binding, we speculate that protein flexibility enables
TRAP to respond to intracellular tryptophan
levels by facilitating access of the allosteric
ligand to its binding site. As the concentration of
tryptophan increases and the binding sites
become populated, many of the disordered
residues fold into place and much of the conformational exchange dynamics is dampened.
Once a sufficient number of the available tryptophan-binding sites are populated, the RNAbinding scaffold is stabilized, generating the
activated protein (Figure 8(b)), which binds to its
cognate RNA with a highly favorable Kd of
10210 M (Figure 8(c)).32
Further, we propose that retaining loop motion
in activated TRAP in the absence and in the
presence of RNA is critical from a regulatory
standpoint, as it may be important to enable facile
exit of tryptophan and de-activation of TRAP in
response to changes in ligand concentration.
Indeed, the rate-limiting step for release of the
RNA has been shown to be tryptophan dissociation.32 Line-broadening of resonances from
the bB – bC loop in the presence of tryptophan
(Figures 5(a) and 6(b)), and the fact that such flexibility would provide a plausible channel between
binding site and solvent (Figure 2(b)) are both
consistent with this proposal. Thus, once the levels
of cellular tryptophan drop sufficiently, the ligandbinding sites become largely unpopulated, the
RNA-binding scaffold becomes too fluid to sup-
TROSY-NMR of Free and Tryptophan-bound TRAP
port RNA binding, and consequently, the 50 leader
folds into the more stable anti-terminator hairpin
structure and attenuation no longer occurs.
Protein dynamics in ligand-regulated
allosteric proteins
Even for the best-characterized allosteric transcriptional regulators, the precise mechanism of
ligand-mediated activation has been difficult to
ascertain with certainty. Crystallographic studies
of the ligand-free and -bound trp16 and lac33 – 35
repressors, for instance, led to proposals that in
each case the ligand induces a conformational
transition (switch) altering the structure/orientation of the DNA-binding motif. However, solution NMR and hydrogen–deuterium exchange
experiments19 – 22 suggest a different picture, in
which ligand binding alters protein dynamics,
thereby perturbing the thermodynamics of the
protein– DNA interaction.36,37 Indeed, it has been
argued on statistical thermodynamic grounds
that dynamics and allostery are linked
inextricably.38 – 40
Although more detailed dynamic and thermodynamic investigations are warranted, the
observed temperature-dependence of tryptophan
binding and accompanying protein rigidification
suggest that the energetic cost of forming an
ordered RNA-binding scaffold is compensated for
by entropy gained in the release of solvent upon
tryptophan binding (i.e. via the hydrophobic
effect). If this interpretation proves to be correct,
TRAP represents an interesting addition to the
emerging theme of folding-coupled nucleic acid
recognition.41,42 The distinction is that in this case
the folding event is coupled to ligand binding as
opposed to nucleic acid binding. Finally, as a
regulatory mechanism, ligand control of protein
flexibility may provide TRAP with the dynamic
(and thermodynamic) balance to achieve the
desired “tuning” of the regulatory switch. That is,
while retaining protein dynamics would allow the
regulatory switch to be readily turned on or off,
once tryptophan has bound and the signal is on,
the unequivocal result is specific, high-affinity
RNA binding. Clearly, more work is needed to
shed light on these interesting mechanistic
hypotheses.
This work highlights the importance of understanding the role of flexibility in regulating protein
function, and reminds us of the complex interplay
of various dynamic and thermodynamic parameters that govern protein structure and ligand
recognition. It is also a demonstration of the critical role that TROSY-based NMR spectroscopy
techniques24 – 27 can play in providing answers to
similar mechanistic questions on macromolecular
complexes of this size.
471
TROSY-NMR of Free and Tryptophan-bound TRAP
Materials and Methods
Preparation of uniformly 2H,13C,15N-labeled apoB. stearothermophilus TRAP
The gene encoding B. stearothermophilus TRAP was
subcloned into pET17b (Novagen, Inc.) and the resulting
plasmid was transformed into E. coli BL21(DE3) cells.
Uniformly isotopically labeled TRAP for NMR experiments was prepared by growing transformed cells in
modified M9 minimal medium containing 50 mg/ml of
ampicillin and 1.0 g/l of 15NH4Cl, 2 g/l of [13C]glucose
and 100% or 75% 2H2O. In this medium, the doubling
time of the cells was approximately one hour during the
logarithmic phase of growth. Expression of TRAP was
induced by addition of IPTG to 1 mM and allowed to
proceed for four to 12 hours. Cells were harvested by
centrifugation, resuspended in 100 mM K2HPO4 (pH
8.0), 50 mM KCl, 1 mM EDTA and broken in a French
pressure cell at 1200 psi (1 psi <6.9 kPa). The lysate was
cleared by centrifugation at 20,000g for 20 minutes and
the supernatant was incubated at 70 8C for 20 minutes
followed by centrifugation at 20,000g for 20 minutes.
The supernatant was applied to a Q-Sepharose ionexchange column equilibrated with 50 mM Tris-HCl
(pH 8.0). TRAP is found in the flow-through from this
column, whereas the remaining contaminating E. coli
proteins are bound. TRAP purified by this method
is over 95% pure as determined by SDS-PAGE. The
yield of uniformly labeled TRAP from this system was
20 – 25 mg/liter.
To remove the tryptophan that co-purifies with the
protein, TRAP was denatured by dialysis for 12 hours at
room temperature in a 3500 Da cutoff dialysis cassette
(Pierce) against 200 ml of 50 mM sodium phosphate (pH
8.0), 100 mM NaCl, 6 M guanidine hydrochloride
(GdnHCl). After 12 hours, the dialysis buffer was diluted
to 3 M GdnHCl with 200 ml of buffer A (50 mM sodium
phosphate (pH 8.0), 100 mM NaCl) and dialyzed for an
additional 12 hours at room temperature. The dialysis
buffer was again diluted to 1.5 M GdnHCl by an adding
400 ml of buffer A and dialyzed for 12 hours at
310 K. The dialysis buffer was then exchanged twice
with 2 l of buffer A and dialyzed for 12 hours each at
310 K. TRAP renatured by this approach is properly
folded as measured by NMR and circular dichroism
spectroscopy, adopts its normal oligomeric state as
assayed by ultra-centrifugation, fluorescence anisotropy
and gel-filtration, and is fully functional for RNA
binding and tryptophan binding.14
NMR sample preparation
The purified protein samples were dialyzed twice for
12 hours at 310 K in a 3500 Da cutoff dialysis cassette
(Pierce) against 2 l of buffer A, followed by addition of
2
H2O to 10% (v/v) and NaN3 to 0.02% (w/v).
NMR spectroscopy
All heteronuclear NMR spectra were collected using
water flip-back, gradient coherence selection and sensitivity enhancement.43 To ascertain the effect of temperature on TRAP, 2D 15N HSQC and TROSY spectra24,28,29
were recorded on a Bruker DMX-600 spectrometer at
5 K intervals from 298 to 345 K. All other data were
recorded on a Bruker DRX-800 at 328 K. HN,N,Ca, and
Cb chemical shift assignments were obtained from
3D TROSY-HNCA and TROSY-HNCACB spectra25,27
(C.M., A.M., A.W., P.G. & M.P.F., unpublished results).
Substantial chemical shift differences between apo and
holo-TRAP required de novo resonance assignment of the
apo-TRAP. The 2D 15N-edited spectra were recorded
with spectral widths of (3333 £ 12,500 Hz) and (64 £
1024) complex points using eight transients/FID in (t1,
t2). NMR data were processed using NMRPipe44 and
analyzed with NMRView.45 1H line-widths in TROSY
spectra were measured with NMRDraw.44 Weightedaverage chemical shift differences, Dav(HNCaCb), were
calculated for the 1H, 15N, and 13Ca, resonances, using:
Dav ðHNCa Þ ¼ ½ðDH2 þ ðDN=5Þ2 þ ðDCa =2Þ2 Þ=31=2
where DH, DN and DCa are the differences between the
chemical shifts in apo and holo-TRAP.46 – 48 Shortestapproach intermolecular distances were obtained by
measuring intermolecular distances and extracting the
shortest distance for each residue from the resulting distance matrix. Data were mapped to the crystal structure
of TRAP in ternary complex with tryptophan and RNA
(1C9S)8 using MOLMOL.49
Proteolytic cleavage
Two aliquots of apo-TRAP (3.4 mg/ml in buffer A,
prepared as described above) were used to examine the
protein’s proteolytic sensitivity. One aliquot was preincubated (for five minutes) with equimolar tryptophan
and both solutions were treated with 5% (w/w) trypsin;
cleavage was assessed at various time intervals by
quenching with 1 mM PMSF, 0.1% (v/v) trifluoroacetic
acid and analyzed by Tris-tricine SDS-10 – 20% PAGE
analysis and reverse-phase HPLC purification (Vydac
218TP54 0.5 cm £ 30 cm C18 column, 45 ml gradient of
20%– 60% (v/v) acetonitrile in 0.1% trifluoroacetic acid,
1 ml/minute, detection at 220 nm) followed by electrospray mass spectrometry in 50% acetonitrile.
Acknowledgements
The authors thank the staff of the OSU Campus
Chemical Instrumentation Center for technical
assistance, A. G. Palmer and P. Loria for pulse
sequences. This work was supported by a
Structural Biology supplement from the National
Institutes of Health, GM62750-01.
References
1. Kuroda, M. I., Henner, D. & Yanofsky, C. (1988). Cisacting sites in the transcript of the Bacillus subtilis trp
operon regulate expression of the operon. J. Bacteriol.
170, 3080– 3088.
2. Hoffman, R. J. & Gollnick, P. (1995). The mtrB gene of
Bacillus pumilus encodes a protein with sequence and
functional homology to the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis. J. Bacteriol.
177, 839– 842.
3. Gollnick, P. (1994). Regulation of the Bacillus subtilis
trp operon by an RNA-binding protein. Mol. Microbiol. 11, 991– 997.
472
4. Babitzke, P. (1997). Regulation of tryptophan biosynthesis: Trp-ing the TRAP or how Bacillus subtilis
reinvented the wheel. Mol. Microbiol. 26, 1– 9.
5. Chen, X., Antson, A. A., Yang, M., Li, P., Baumann,
C., Dodson, E. J. et al. (1999). Regulatory features of
the trp operon and the crystal structure of the trp
RNA-binding attenuation protein from Bacillus
stearothermophilus. J. Mol. Biol. 289, 1003 –1016.
6. Otridge, J. & Gollnick, P. (1993). MtrB from Bacillus
subtilis binds specifically to trp leader RNA in a
tryptophan-dependent manner. Proc. Natl Acad. Sci.
USA, 90, 128– 132.
7. Antson, A. A., Otridge, J., Brzozowski, A. M.,
Dodson, E. J., Dodson, G. G., Wilson, K. S. et al.
(1995). The structure of trp RNA-binding attenuation
protein. Nature, 374, 693– 700.
8. Antson, A. A., Dodson, E. J., Dodson, G., Greaves,
R. B., Chen, X. & Gollnick, P. (1999). Structure of the
trp RNA-binding attenuation protein, TRAP, bound
to RNA. Nature, 401, 235– 242.
9. Steegborn, C., Danot, O., Huber, R. & Clausen, T.
(2001). Crystal structure of transcription factor MalT
domain III: a novel helix repeat fold implicated in
regulated oligomerization. Structure, 9, 1051– 1060.
10. Schreiber, V. & Richet, E. (1999). Self-association of
the Escherichia coli transcription activator MalT in
the presence of maltotriose and ATP. J. Biol. Chem.
274, 33220– 33226.
11. Zhu, J. & Winans, S. C. (2001). The quorum-sensing
transcriptional regulator TraR requires its cognate
signaling ligand for protein folding, protease resistance, and dimerization. Proc. Natl Acad. Sci. USA, 98,
1507 –1512.
12. Zhang, R. G., Pappas, T., Brace, J. L., Miller, P. C.,
Oulmassov, T., Molyneaux, J. M. et al. (2002).
Structure of a bacterial quorum-sensing transcription
factor complexed with pheromone and DNA. Nature,
417, 971– 974.
13. Depoix, C., Delmotte, M. H., Formstecher, P. &
Lefebvre, P. (2001). Control of retinoic acid receptor
heterodimerization by ligand-induced structural
transitions. A novel mechanism of action for retinoid
antagonists. J. Biol. Chem. 276, 9452– 9459.
14. Li, P. T., Scott, D. & Gollnick, P. (2002). Creating
hetero-11-mers composed of wild-type and mutant
subunits to study RNA binding to TRAP. J. Biol.
Chem. 227, 11838– 11844.
15. Passner, J. M., Schultz, S. C. & Steitz, T. A. (2000).
Modeling the cAMP-induced allosteric transition
using the crystal structure of CAP-cAMP at 2.1 Å
resolution. J. Mol. Biol. 304, 847– 859.
16. Otwinowski, Z., Schevitz, R. W., Zhang, R. G.,
Lawson, C. L., Joachimiak, A., Marmorstein, R. Q.
et al. (1988). Crystal structure of trp repressor/
operator complex at atomic resolution. Nature, 335,
321– 329.
17. Yang, N., Schule, R., Mangelsdorf, D. J. & Evans,
R. M. (1991). Characterization of DNA binding and
retinoic acid binding properties of retinoic acid
receptor. Proc. Natl Acad. Sci. USA, 88, 3559– 3563.
18. Egea, P. F., Mitschler, A., Rochel, N., Ruff, M.,
Chambon, P. & Moras, D. (2000). Crystal structure of
the human RXRalpha ligand-binding domain bound
to its natural ligand: 9-cis retinoic acid. EMBO J. 19,
2592– 2601.
19. Kalodimos, C. G., Boelens, R. & Kaptein, R. (2002). A
residue-specific view of the association and dissociation pathway in protein– DNA recognition.
Nature Struct. Biol. 9, 193– 197.
TROSY-NMR of Free and Tryptophan-bound TRAP
20. Arrowsmith, C. H., Czaplicki, J., Iyer, S. B. &
Jardetzky, O. (1991). Unusual dynamic features of
the trp repressor from Escherichia coli. J. Am. Chem.
Soc. 113, 4020– 4022.
21. Arrowsmith, C., Pachter, R., Altman, R. & Jardetzky,
O. (1991). The solution structures of Escherichia coli
trp repressor and trp aporepressor at an intermediate
resolution. Eur. J. Biochem. 202, 53 – 66.
22. Zhao, D., Arrowsmith, C. H., Jia, X. & Jardetzky, O.
(1993). Refined solution structures of the Escherichia
coli trp holo- and aporepressor. J. Mol. Biol. 229,
735– 746.
23. van Tilborg, P. J., Czisch, M., Mulder, F. A., Folkers,
G. E., Bonvin, A. M., Nair, M. et al. (2000). Changes
in dynamical behavior of the retinoid X receptor
DNA-binding domain upon binding to a 14 basepair DNA half site. Biochemistry, 39, 8747– 8757.
24. Pervushin, K., Riek, R., Wider, G. & Wuthrich, K.
(1997). Attenuated T2 relaxation by mutual cancellation of dipole – dipole coupling and chemical shift
anisotropy indicates an avenue to NMR structures
of very large biological macromolecules in solution.
Proc. Natl Acad. Sci. USA, 94, 12366 –12371.
25. Salzmann, M., Pervushin, K., Wider, G., Senn, H. &
Wuthrich, K. (1998). TROSY in triple-resonance
experiments: new perspectives for sequential NMR
assignment of large proteins. Proc. Natl Acad. Sci.
USA, 95, 13585– 13590.
26. Salzmann, M., Wider, G., Pervushin, K. & Wuthrich,
K. (1999). Improved sensitivity and coherence selection for [15N,1H]-TROSY elements in triple resonance
experiments. J. Biomol. NMR, 15, 181– 184.
27. Salzmann, M., Pervushin, K., Wider, G., Senn, H. &
Wuthrich, K. (1999). [13C]-constant-time [15N,1H]TROSY-HNCA for sequential assignments of large
proteins. J. Biomol. NMR, 14, 85 – 88.
28. Rance, M., Loria, J. P. & Palmer, A. G. r. (1999).
Sensitivity improvement of transverse relaxationoptimized spectroscopy. J. Magn. Reson. 136, 92– 101.
29. Loria, J. P., Rance, M. & Palmer, A. G., III (1999).
Transverse-relaxation-optimized (TROSY) gradientenhanced triple-resonance NMR spectroscopy.
J. Magn. Reson. 141, 180– 184.
30. Ramesh, V. & Brown, T. (1996). 1H-NMR characterization of L -tryptophan binding to TRAP, the trp
RNA-binding attenuation protein of Bacillus subtilis.
Biochem. J. 315, 895– 900.
31. Yang, M., Chen, X., Militello, K., Hoffman, R.,
Fernandez, B., Baumann, C. & Gollnick, P. (1997).
Alanine-scanning mutagenesis of Bacillus subtilis trp
RNA-binding attenuation protein (TRAP) reveals
residues involved in tryptophan binding and RNA
binding. J. Mol. Biol. 270, 696– 710.
32. Baumann, C., Otridge, J. & Gollnick, P. (1996). Kinetic
and thermodynamic analysis of the interaction
between TRAP (trp RNA-binding attenuation protein) of Bacillus subtilis and trp leader RNA. J. Biol.
Chem. 271, 12269– 12274.
33. Lewis, M., Chang, G., Horton, N. C., Kercher, M. A.,
Pace, H. C., Schumacher, M. A. et al. (1996). Crystal
structure of the lactose operon repressor and its
complexes with DNA and inducer. Science, 271,
1247– 1254.
34. Bell, C. E. & Lewis, M. (2001). The Lac repressor:
a second generation of structural and functional
studies. Curr. Opin. Struct. Biol. 11, 19 – 25.
35. Bell, C. E. & Lewis, M. (2000). A closer view of the
conformation of the Lac repressor bound to operator.
Nature Struct. Biol. 7, 209– 214.
473
TROSY-NMR of Free and Tryptophan-bound TRAP
36. Perutz, M. F. (1989). Mechanisms of cooperativity
and allosteric regulation in proteins. Quart. Rev.
Biophys. 22, 139– 237.
37. Jardetzky, O. (1996). Protein dynamics and conformational transitions in allosteric proteins. Prog. Biophys. Mol. Biol. 65, 171– 219.
38. Luque, I. & Freire, E. (2000). Structural stability of
binding sites: consequences for binding affinity and
allosteric effects. Proteins: Struct. Funct. Genet., 63 – 71.
39. Sturtevant, J. M. (1977). Heat capacity and entropy
changes in processes involving proteins. Proc. Natl
Acad. Sci. USA, 74, 2236– 2240.
40. Cooper, A. & Dryden, D. T. (1984). Allostery without
conformational change. A plausible model. Eur. Biophys. J. 11, 103–109.
41. Spolar, R. S. & Record, M. T., Jr (1994). Coupling of
local folding to site-specific binding of proteins to
DNA. Science, 263, 777– 784.
42. Williamson, J. R. (2000). Induced fit in RNA – protein
recognition. Nature Struct. Biol. 7, 834– 837.
43. Sattler, M., Schleucher, J. & Griesinger, C. (1999).
Heteronuclear multidimensional NMR experiments
for the structure determination of proteins in solution employing pulsed field gradients. Prog. NMR
Spectrosc. 34, 93 – 158.
44. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G.,
Pfeifer, J. & Bax, A. (1995). NMRPipe: a multi-
45.
46.
47.
48.
49.
dimensional spectral processing system based on
UNIX pipes. J. Biomol. NMR, 6, 277– 293.
Johnson, B. & Blevins, R. (1994). NMR View—a computer-program for the visualization and analysis of
NMR data. J. Biomol. NMR, 4, 603– 614.
Foster, M. P., Wuttke, D. S., Clemens, K. R., Jahnke,
W., Radhakrishnan, I., Tennant, L. et al. (1998).
Chemical shift as a probe of molecular interfaces:
NMR studies of DNA binding by the three aminoterminal zinc finger domains from transcription
factor IIIA. J. Biomol. NMR, 12, 51 – 71.
Garrett, D. S., Seok, Y. J., Peterkofsky, A., Clore, G. M.
& Gronenborn, A. M. (1997). Identification by NMR
of the binding surface for the histidine-containing
phosphocarrier protein HPr on the N-terminal
domain of enzyme I of the Escherichia coli phosphotransferase system. Biochemistry, 36, 4393– 4398.
Grzesiek, S., Bax, A., Clore, G. M., Gronenborn, A. M.,
Hu, J. S., Kaufman, J. et al. (1996). The solution structure of HIV-1 Nef reveals an unexpected fold and
permits delineation of the binding surface for the
SH3 domain of Hck tyrosine protein kinase. Nature
Struct. Biol. 3, 340– 345.
Koradi, R., Billeter, M. & Wuthrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 51-55, 29 – 32.
Edited by M. F. Summers
(Received 12 June 2002; received in revised form 27 August 2002; accepted 27 August 2002)