From the Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany
Received for publication, March 14, 2001
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ABSTRACT |
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The Drosophila Bicoid (Bcd)
protein plays a dual role as a transcription and translation factor
dependent on the unique DNA and RNA binding properties of the
homeodomain (HD). We have used circular dichroism and
fluorescence spectroscopy to probe the structure and stability of the
Bcd-HD, for which a high resolution structure is not yet available. The
fluorescence from the single tryptophan residue in the HD (Trp-48) is
strongly quenched in the native state but is dramatically enhanced
(~20-fold) upon denaturation. Similar results were obtained with the
Ultrabithorax HD (Ubx-HD), suggesting that the unusual tryptophan
fluorescence may be a general phenomenon of HD proteins. We have used
site-directed mutagenesis to explore the role of aromatic acids in the
structure of the Bcd-HD and to evaluate the proposal that interactions
between the strictly conserved Trp residue in HDs and nearby aromatic residues are responsible for the fluorescence quenching in the native
state. We determined that both Trp-48 and Phe-8 in the N-terminal
region of the HD are individually necessary for structural stability of the Bcd-HD, the latter most likely as a factor
coordinating the orientation of the N-terminal helix I and the
recognition helix for efficient binding to a DNA target.
The regulation of gene expression is generally accomplished
by interactions of proteins with nucleic acids. These regulators contain DNA binding domains (transcription factors) or RNA binding domains (translational regulators). The homeobox encodes a 60-amino acid nucleic acid binding domain, the homeodomain
(HD),1 which is present in
many eukaryotic transcription factors and plays a critical role in
development (1, 2). The sequences have been well conserved in the
course of evolution from fungi to vertebrates. In addition to binding
DNA, thereby activating transcription, the Drosophila Bcd-HD
has been recently shown to interact with RNA (3-5). Thus, Bcd has a
dual role in development, directing pattern formation along the
anterior-posterior axis of the embryo by cooperative DNA binding and
subsequent gene activation (6-8) and by binding the mRNA of the
regulatory gene caudal (cad), thereby repressing
its translation (3, 5). Because of this unique DNA and RNA binding
capability, Bcd-HD is a particularly compelling target for
investigations of molecular structure and nucleic acid recognition mechanisms.
The HD has also become a paradigm for studying DNA-protein
interactions. HDs fold into a module with a characteristic structure comprising three We have used biophysical methods combined with site-directed
mutagenesis to elucidate the molecular structure-function relationships of the Bcd-HD, for which a high resolution structure is not yet available and have focused on the highly conserved aromatic residues tryptophan (Trp-48, helix III), and phenylalanine (Phe-8, helix I). In
homeodomains, Trp-48 exhibits a strongly quenched fluorescence in the
native state (22-24) most likely because of specific interactions with
neighboring residues. Conservative mutation of the strictly conserved
Trp-48 to a phenylalanine (W48F) reduces DNA binding affinity and
destabilizes the HD, suggesting that the role of Trp-48 is to stabilize
critical structural features required for DNA recognition. Replacement
of Phe-8 by a tyrosine residue (F8Y, Antennapedia like) yields a stable
HD with fluorescence and binding properties similar to the wild-type
HD. Replacement of Phe-8 by an alanine (F8A) yields a less stable HD,
with lower quenching of Trp-48 fluorescence in the native state. These
results suggest the following. (i) An aromatic residue in position 8 may be required to provide the scaffold, holding the HD in a
conformation required for DNA binding and (ii) that the quenching of
Trp-48 in the native state has more complex origins.
Protein Expression and Purification--
The recombinant protein
(Bcd-HD) was expressed and purified as described (8). Protein purity
was confirmed by SDS-polyacrylamide gel electrophoresis. Further
purification was performed as required over an Amersham Pharmacia
Biotech mono-P column in 10 mM triethanolamine-HCl buffer,
pH 7.0. Site-directed mutagenesis of Bcd-HD was carried out using
the Stratagene QuikChange mutagenesis kit using the following primers
and the wild-type Bcd-HD plasmid pRSETBcdHDCHis (8) as a template:
W48F, 5'-GCCCAGGTGAAGATATTTTTTAAGAACCGTCGGCGTC-3' and
5'-GACGCCGACGGTTCTTAAAAAATATCTTCACCTGGGC-3'; F8Y,
5'-CGTCGCACCCGCACCACTTATACCAGCTCTCAAATAG-3' and
5'-CTATTTGAGAGCTGGTATAAGTGGTGCGGGTGCGACG-3'; F8A,
5'-CGTCGCACCCGCACCACTGCTACCAGCTCTCAAATAG and
5'-CTATTTGAGAGCTGGTAGCAGTGGTGCGGGTGCGACG-3'. Expression and purification of the mutant proteins (Bcd-HD W48F, F8Y, and F8A) was
performed as described (8). The proteins were extensively dialyzed
against 10 mM sodium phosphate buffer, pH 7.0, 50 mM NaCl for fluorescence and CD experiments.
CD Spectroscopy--
CD measurements were performed on a JASCO
J-720 CD spectropolarimeter. Far-UV CD data are presented as molecular
ellipticity [ Steady-State Fluorescence--
Fluorescence was measured on an
SLM 8000C (SLM, Urbana, IL) or a PTI Quantamaster (Photon Technology
International, Lawrenceville, NJ) photon counting spectrofluorometer
with double excitation and single emission monochromators. The
excitation wavelength was 280 nm or 295 nm (to avoid excitation of Tyr
and Phe). Polarization effects were eliminated by using magic angle
settings of the polarizers (0° excitation, 54.7° emission). The
emission spectra were corrected, and a solvent blank was subtracted.
All measurements were at 5 °C.
Time-resolved Fluorescence--
Time-resolved measurements
were performed in the frequency domain using a system of our design
incorporating a modulated D2 lamp (Cathodeon, Cambridge,
UK) as the excitation source (26, 27). All measurements were at
5 °C. Excitation at 280 nm was through a 10-nm bandpass filter, and
the emission was selected with a 325-nm long-pass filter. The reference
lifetime compound was N-acetyl-tryptophanamide in 30% (v/v)
glycerol/water solution, which has a lifetime of 4.88 ns at 5 °C
(28). The data were analyzed using global analysis software (Globals
Unlimited, LFD, Urbana, IL).
In Vitro Binding Assays--
The Bcd target site
oligonucleotides (5'-AATCTAATCCCTATA-3') were
5'- The Bcd-HD Has Substantial Helical Structure--
The CD
spectra of the Bcd-HD under native conditions showed a double minimum
at 208 and 222 nm, characteristic of a helical structure (Fig.
1a), and similar to that
observed for other homeodomains (21, 30, 31). A secondary structure
prediction algorithm applied to the spectra yielded an Trp Fluorescence Is Strongly Quenched in the Native State of
the Bcd-HD and Ubx-HD--
We further explored the structure of the
Bcd-HD using the fluorescence of Trp-48. Trp-48 was excited selectively
at 295 nm to avoid contributions from the single Tyr residue in the
sequence, Tyr-25. In the folded state, the emission was strongly
quenched (95%) relative to that in the unfolded state (Fig.
2a). The protein fluorescence
excited at 280 nm was dominated by Tyr-25 and had an emission peak at
314 nm (Fig. 2b). Upon unfolding in 6 M
guanidinium hydrochloride (GdmHCl), the intensity of the Trp-48
fluorescence (excited at 295 nm) increased by a factor of ~20 and was
accompanied by a 15-nm red-shift in the emission spectra (Fig.
2c). A similar result was obtained for unfolding in urea
(data not shown). For comparison, we studied the fluorescence
properties of the Ubx-HD. This HD also contains a single Trp (Trp-48)
but unlike Bcd-HD has three Tyr residues (Tyr-8, Tyr-11, and Tyr-25).
As in the Bcd-HD, the Trp-48 fluorescence of the Ubx-HD in the native
state was strongly quenched (Fig. 2d). This result suggests
that the quenching of Trp-48 fluorescence is not restricted to the
Bcd-HD but may rather be a property of homeodomains in general,
implying that Trp-48 may serve as a sensitive conformational marker for studying the entire homeodomain protein family. Similar results have
been observed for other homeodomains by Nanda and Brand (23).
The quenching of Trp was explored further by measuring the
fluorescence lifetimes of the Bcd and Ubx HDs in the native and GdmHCl-denatured states, using frequency domain fluorimetry. In both
cases, the native HDs exhibited shorter fluorescence lifetimes than the
denatured HDs, evidenced by the higher frequency crossovers of the
phase and modulation curves (data not shown). The fluorescence decays
were multicomponent, yielding mean lifetimes of ~4.1 ns for native
Bcd-HD, ~4.9 ns for denatured Bcd-HD, ~1.9 ns for native Ubx-HD,
and ~5.1 ns for denatured Ubx-HD. The ratio of the native and
denatured lifetimes did not correspond to the relative quantum yields,
indicating a combination of dynamic and static quenching mechanisms. We
note that both native and denatured lifetimes were multi-exponential
and that the analysis of the native state fluorescence lifetime was
difficult because of the potential contamination by even a minute
fraction of denatured or partially denatured polypeptide, leading to a
perturbation of the mean lifetime to longer values. Nonetheless, the
trend (reduction of fluorescence lifetime, i.e. quantum
yield, in the native state) was clear in both cases.
Mutation of Trp-48 Abolishes DNA Binding--
We mutated
Trp-48 in the recognition helix to a Phe to evaluate the role of this
residue in determining the structure and binding properties of the
Bcd-HD. Phe was selected as a conservative replacement for Trp-48 to
avoid disrupting the immediate environment of helix III. The
equilibrium CD spectra of Bcd-HD W48F revealed the characteristic
helical minima at 208 and 222 nm (Fig. 1a), with a predicted
helical content of ~35% (Fig. 1a, inset),
suggesting that the mutation did not have significant effects at the
level of secondary structure. However, electrophoretic mobility shift assays showed that Bcd-HD W48F bound DNA with significantly reduced affinity compared with the wild-type Bcd-HD (Fig.
3; a, b and e). This result was not consistent with the limited effect
on secondary structure. Furthermore, structural data based on x-ray crystallography of HDs had indicated that Trp-48 is not critically involved in DNA binding, providing only indirect contacts to the phosphate backbone. It follows that the pronounced reduction of affinity for DNA could not be attributed solely to a slight change in
secondary structure or loss of a weak contact to DNA.
To further investigate the origin of the impaired DNA binding of Bcd-HD
W48F, we analyzed the stability of the mutated HD. The thermal
denaturation of Bcd-HD W48F monitored by CD spectroscopy revealed a
sharp reduction in the Tm from 44 to 27 °C
(Fig. 4), suggesting that the mutation
had a strong destabilizing effect. We conclude that Trp-48 imparts
rigidity to the helix-turn-helix motif of the homeodomain, perhaps
thereby potentiating DNA binding.
As expected, the Bcd-HD W48F exhibited a fluorescence spectrum
characteristic of the single Tyr-25 in the mutant protein. Upon
unfolding Bcd-HD W48F in GdmHCl or urea, the fluorescence intensity
increased by ~50% (data not shown).
An Aromatic Residue in Position 8 Is Required for Stability of the
HD--
We mutated the native phenylalanine residue in position 8 of
the HD to an alanine or to a tyrosine (similar to that of the Antennapedia HD, which is often described as the canonical HD). Both
mutants exhibited significant helical structure measured by CD (data
not shown). From the electrophoretic mobility shift assay, Bcd-HD F8A
had a similarly reduced affinity to DNA as Bcd-HD W48F, whereas
the Antennapedia-like Bcd-HD F8Y mutant retained the wild-type binding
affinity (Fig. 3, c-e). The Bcd-HD F8A mutant was destabilized
(Tm 37 °C) whereas the Bcd-HD F8Y mutant is
slightly more stable (Tm 46 °C) than the
wild-type HD (Tm 44 °C, see also Fig. 4).
Fluorescence spectra of native and denatured Bcd-HD F8A and Bcd-HD F8Y
mutants are shown in Fig. 2, e and f. The Trp
fluorescence intensity of denatured Bcd-HD F8A was ~11 times that of
the native Bcd-HD F8A, whereas denatured Bcd-HD F8Y showed an intensity
~23 times larger than the native Bcd-HD F8Y. Clearly there was a
correlation between the presence of an aromatic residue in position 8 of the HD and the quenching of native Trp fluorescence, but this
interaction could not account for the full extent of the quenching.
Molecular Modeling of Bcd-HD Structure--
Knowledge-based
protein modeling methods (SWISS-MODEL automated protein modeling server
(35, 36)) were used to generate a model for the structure of the
Bcd-HD (Fig. 5, a and
b) based on the coordinates of existing homeodomain
structures. The relative orientations of the three helices and the
positions of Trp-48 and other aromatic residues (Phe-8, Phe-20, Tyr-25,
Phe-49) in the Bcd-HD are shown in this model.
We have probed the structural characteristics of Bcd-HD, a nucleic
acid binding domain specifically recognizing RNA and DNA in its
capacity as a dual regulator of gene expression and translation, by
exploiting the single Trp in the molecule (Trp-48), a strictly invariant residue in the primary sequences of the HD family, as a
sensitive probe of conformational changes in the protein. The strict
conservation of Trp-48 in the HD family suggests that it plays a key
role in the function of homeodomains. Although Trp-48 lies in the
recognition helix, it is not involved in direct contacts with the DNA
but rather makes a water-mediated hydrogen bond with the phosphate
backbone (12, 15, 37).
Structural Aspects--
The CD spectra confirmed that the
Trp-48 Fluorescence Properties--
According to the relatively
blue-shifted fluorescence peak (329 nm) in the native state, Trp-48 is
buried within the molecule and thus shielded from solvent. The few
known HD structures and molecular modeling of the Bcd-HD are consistent
with these data. The fluorescence of Trp-48 increased dramatically
(20-fold) upon denaturation. That is, Trp-48 fluorescence displays an
unprecedented degree of quenching in the native state. Ubx-HD also
possesses a single Trp residue that is highly quenched (this work and
Ref. 23). In view of the observations of Nanda and Brand (23, 24) of
native state Trp quenching in the Ubx, Engrailed, and Antennapedia C39S
mutant HDs, we conclude that the fluorescence properties of Trp-48 may
be general in the context of the HD fold and thus applicable to all
HDs. In other words, Trp-48 fluorescence may be useful as a probe for
comparing the structures of other members of this family for which high
resolution structural information is not yet available.
Role of Phe-8--
A surprising result of this work is the
importance of the aromatic residue in position 8. The Bcd-HD F8A mutant
shows a significantly reduced DNA binding ability (comparable with
W48F) and a destabilized structure. Conversely, the Bcd-HD F8Y mutant
binds as well as the wild-type HD and is slightly more stable. This
suggests that an aromatic residue in position 8, and the resultant
interaction with Trp-48, may be critical to mediate the structure of
the HD by bringing helix I and the recognition helix, helix III, into a
conformation optimal for DNA binding.
Mechanisms of Trp-48 Fluorescence Quenching--
Carra and
Privalov (30) reported quenching of fluorescence in the native state of
the MAT
In the absence of a x-ray crystallographic or solution
NMR-derived structure for the Bcd-HD, one cannot state conclusively that the proposed interaction plays a role in the quenching of the
Trp-48 in the Bcd-HD. Closer inspection of the surroundings of Trp-48
in the homology-based molecular model of the Bcd-HD reveal a possible
interaction of the indole nitrogen with the benzene ring of Phe-8 (Fig.
5b; ~ 4 Å distance of closest approach), which might
contribute to the quenching of Trp-48. In fact the planes of the
aromatic rings of the two residues are almost orthogonal to each other
(Fig. 5b). Similarly, although a high-resolution structure
of the Ubx-HD is not available, it is plausible that an interaction
between Trp-48 and the aromatic ring of Tyr-8 in Ubx-HD also
contributes to the fluorescence quenching. A survey of the known
homeodomains reveals that all, except for the LIM and POU class
proteins, possess a Tyr or Phe residue at position 8. However, as our
fluorescence data for the Bcd-HD F8A and Bcd-HD F8Y mutants (Fig. 2,
e-f) indicate, this interaction alone cannot account for
the full extent of the quenching. That is, other mechanisms or
interactions must intervene, possibly involving other neighboring aromatic residues, which are clustered around Trp-48 (Fig.
5b). A likely candidate is Phe-49, which is also nearly
invariant in HDs with the exception of the MAT
Chen and Barkley (45) have recently identified amino
acid side chains that quench Trp fluorescence in solution. Lys and Tyr residues quench by excited-state proton transfer whereas Gln, Asn,
Glu, Asp, Cys, and His quench by electron transfer. Electron transfer
has less stringent geometric requirements than proton transfer, can
occur through-space or through-bond over distances of up to 10 Å, and
is thus likely to play a major role in quenching. In the Bcd model
structure described above, His-19 and His-56 are within 10 Å of
Trp-48. However the ionization state of His residues critically affects
their quenching ability, with positively charged His residues being
much more effective than neutral His residues. We would not expect
His-19 or His-56 to be protonated under our experimental conditions.
The quenching ability of other amino acid side-chains such as Arg, of
which a stretch of four residues occurs in helix III of the Bcd HD,
might be enhanced by the proximity and orientation of these side-chains
in the context of the protein fold, even though at neutral pH these
show negligible quenching of model compound fluorescence (45).
Further insight into the dynamics and nature of the interactions of the
indole proton of Trp-48 of Bcd-HD with surrounding residues will be
provided by hydrogen/deuterium exchange experiments and by progress in
the structure determination of the Bcd-HD.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices. Structural studies, first elucidated for
the Antennapedia and Engrailed HDs, show that the three helices of the HD are stabilized by hydrophobic interactions involving residues
Leu-16, Trp-48, and Phe-49, which are conserved in all HDs (9-15). The
HD shows a common helix-turn-helix motif encompassing the recognition
helix III, which docks into the major groove of DNA. The specificity of
DNA recognition is achieved by specific amino acid residue-base
contacts in helix III (16), e.g. amino acid residue 50 (glutamine in most HDs, lysine in the case of Bcd-HD) both in
vitro and in vivo (4, 7, 17-21). Despite the extensive
data on sequence and structure-function relationships accumulated over
the past years, some aspects of HD function still remain obscure.
Little is known about how HDs achieve specific recognition of their
targets, the role of the N-terminal arm, and the effects of other
highly conserved residues, mostly within helix III. Among these, the
centrally located Trp residue (Trp-48) is invariant. The available
structural data indicate that Trp-48 plays a role in DNA-protein
interactions through a water-mediated hydrogen bond with the phosphate
backbone (10-14). However, the absolute conservation of Trp-48 in
evolution indicates a far more important function in the HD.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
] (in deg·cm2·dmol
1).
Secondary structure was predicted using the SSE-338 prediction algorithm supplied by JASCO, using the reference spectra of Yang et al. (25).
-32P-labeled with polynucleotide kinase (29). Gel
shift assays were carried out as described (7). Briefly, DNA (10 fmols) and protein were mixed in 25 mM potassium Hepes, pH 7.5, 0.1 M KCl, 12.5 mM MgCl2, 0.1%
Nonidet P-40, 20% glycerol at 4 °C for 15 min. Free and complexed
DNA were resolved by electrophoresis on 12% polyacrylamide gels in
0.5× TBE buffer at 12 V/cm. The DNA concentrations were sufficiently
low so that the fraction of bound protein was negligible. Quantitation
was performed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical
content of 41% (Fig. 1a, inset). As noted by
Ades and Sauer for the Engrailed HD (21), the CD signal at 222 nm was
somewhat lower than that expected for a protein with a ~60% helical
content, but the discrepancy can be attributed to positive ellipticity
contributions from the aromatic residues in the sequence (32, 33). We
monitored the loss of helical structure upon thermal denaturation of
the Bcd-HD by measuring the CD signal at 222 nm as a function of
temperature (Fig. 1b). The protein exhibited a two-state
folding transition with a Tm ~44 °C,
comparable with the value reported for the rat TTF-1 HD (42.8 °C)
(31) and the Antennapedia HD (48 °C) (34). A fit to the thermal
denaturation data yielded a
Hm ~38 kcal mol
1. The unfolding of the Bcd-HD monitored by the Trp-48
fluorescence produced a similar Tm.
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Fig. 1.
CD spectra of the wild-type Bcd-HD and the
Bcd-HD W48F mutant. a, equilibrium CD spectra of
wild-type Bcd-HD (solid line) and Bcd-HD W48F mutant
(solid symbols). Inset, secondary
structure estimation parameters using the algorithm of Yang et
al. (25) for both Bcd-HD and the Bcd-HD W48F mutant. b,
thermal denaturation of Bcd-HD wild type monitored by
[ ]222. Fits to the pre- and post-transition baselines
are shown. Tm ~44 °C.
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Fig. 2.
Fluorescence characterization of Bcd and Ubx
homeodomains. a, native (solid line) and
denatured (6 M GdmHCl) (hyphenated line) Bcd-HD,
10.2 µM. ex = 295 nm. b, native
Bcd-HD.
ex = 280 nm (solid line),
ex = 295 nm (hyphenated line). c,
comparison of native and denatured Bcd-HD fluorescence.
ex = 295 nm. d, native (solid
line) and denatured (6 M GdmHCl) (hyphenated
line) Ubx-HD, 11 µM.
ex = 295 nm.
e, native (solid line) and denatured (6 M GdmHCl) (hyphenated line) Bcd-HD F8A, 4.8 µM.
ex = 295 nm. f, native
(solid line) and denatured (6 M GdmHCl)
(hyphenated line) Bcd-HD F8Y, 8.5 µM.
ex = 295 nm.
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Fig. 3.
Affinity for DNA of the wild type and
W48F, F8A, and F8Y mutants of Bcd-HD. Electrophoretic mobility
shift assay of Bcd-HD wild type (WT; a); Bcd-HD W48F
(b); Bcd-HD F8Y (c); and Bcd-HD F8A
(d) binding to target DNA. The free probe and protein-DNA
complexes are indicated by arrows marked F and
C, respectively. e, quantitation of relative
affinities for DNA. Percent-bound probe is defined as ratio of bound
probe to sum of bound and free probes. WT (closed circle);
Bcd-HD W48F (open circle); Bcd-HD F8Y (open
triangle); Bcd-HD F8A (closed triangle).
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Fig. 4.
Thermal denaturation profiles of Bcd-HD wild
type (WT, curve 3), Bcd-HD F8A
(curve 2), Bcd-HD F8Y(curve 4), and
Bcd-HD W48F (curve 1). Bcd-HD W48F is
significantly destabilized (Tm ~17 °C)
relative to Bcd-HD wild type (Tm
~44 °C).
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Fig. 5.
Knowledge-based structure prediction of
Bcd-HD. a, predicted model for Bcd-HD showing
helices I, II, and III, and positions of the aromatic residues Trp-48,
Tyr-25, and Phe-8. The dashed line indicates a possible
interaction between Trp-48 and Phe-8. b, predicted structure
of residues within 7 Å of Trp-48 in Bcd-HD. The orientations of the
planes of the aromatic rings of Trp-48 and Phe-8 are nearly orthogonal.
The distance of closest approach between the indole NH and Phe-8 is
~4 Å. An atypical hydrogen bond ( -) between these residues may
contribute to the strong fluorescence quenching in Bcd-HD (see
text).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical content of the Bcd-HD is similar to that of other HDs. From
the thermal denaturation profile, the Bcd-HD is an autonomously folded
domain exhibiting a reversible two-state unfolding transition. The
Tm (44 °C) is comparable with that of other
known homeodomains (30, 31). The conservative mutation W48F, designed
to minimally perturb the overall hydrophobicity of the helix III
environment, has a significant
-helical structure yet exhibits
50-fold lower affinity for DNA, suggesting that Trp-48 plays a more
complex role in DNA binding than that postulated previously. In
addition, the dramatic destabilization (reduction of
Tm by ~17°) of the mutant confirms that
Trp-48 forms an integral part of the hydrophobic core of the HD. Trp-48
and Phe-49 are conserved in all known HDs and must play a role in
stabilizing the folded structure and determining the spatial
relationships required for DNA recognition (12). In addition to a
direct role in DNA binding, a major function of Trp-48 in the helix III
of the HD is to stabilize the global structure, thus allowing other
residues to make critical DNA contacts.
2-HD and suggested that interactions with adjacent residues
might be responsible. Native state quenching was also observed in the
trp aporepressor protein (38) and recently in the
immunophilin domain of the FK506-binding protein FKBP59-I (39).
FKBP59-I contains two Trp residues, one of which is deeply buried in
the hydrophobic core and which fluoresces very weakly compared with the
second, surface-exposed, Trp residue. The quenching of this residue was
attributed to an atypical hydrogen-bond interaction involving the
indole nitrogen of the buried Trp and the benzene ring of a neighboring
Phe residue. In this conformation, the indole NH group points toward
the center of the Phe benzene ring, which can act as a hydrogen-bond
acceptor interacting with a donor (40). The strength of this
interaction (~3 kcal mol
1) is about half that of
commonly occurring H-bonds (40) and 5× kT at room
temperature. Thus, it could have a significant role in stabilizing
molecular interactions. Burley and Petsko (41) found that such
interactions between amino and aromatic groups occur more frequently
than expected. In FKBP59-I, the planes of the aromatic rings of the
interacting Trp and Phe residues are oriented almost perpendicular to
each other, which could be expected to facilitate the quenching more
than a parallel configuration. Using time-correlated single photon
counting techniques, Suwaiyan and Klein (42) measured a dramatic
decrease in the lifetime of indole in cyclohexane solution upon the
addition of benzene and suggested that the quenching was because of
H-bonding between the indole NH and the
system of benzene. In
addition, Van Duuren (43) showed that the fluorescence intensities of
1,2-dimethylindole and 1-methyl-2-phenylindole do not decrease in
benzene solution relative to other solvents, implying that the free
indole NH is essential for such complex formation. These results
correlate well with the observed decrease of the lifetime of Trp-48
fluorescence in Bcd-HD and Ubx-HD. Nanda and Brand (23) have used
similar arguments to suggest that the Trp-Phe NH-
hydrogen bond is
responsible for the quenching of the Trp fluorescence in HDs and have
proposed an excited state rearrangement model in which a simple
rotation of the Trp around the C
-C
bond
would be sufficient to bring the indole nitrogen in proximity to the
aromatic acceptor on the N-terminal arm.
2 (valine) and DmBarHI
and DaBarHI (tyrosine) (44).
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ACKNOWLEDGEMENTS |
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We thank Phil Beachy (Johns Hopkins University) for providing us with the purified Ubx-HD. We also thank Gudrun Heim and Annelies Zechel for technical assistance and Gordon Dowe for assistance with DNA sequencing.
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FOOTNOTES |
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* 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.
Recipient of a long term fellowship from the Human Frontiers
Science Program Organization during part of this work.
§ To whom correspondence should be addressed: Tel.: 49 551 2011390; Fax: 49 551 2011467; E-mail: rrivera@gwdg.de.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M102292200
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ABBREVIATIONS |
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The abbreviations used are: HD, homeodomain; Bcd, Bicoid; Ubx, Ultrabithorax; GdmHCl, guanidinium hydrochloride.
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REFERENCES |
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