From the Department of Biochemistry, ¶ McGill
Cancer Centre,
Department of Chemistry, and
** Montreal Joint Centre for Structural Biology, McGill
University, Montreal, Quebec H3G 1Y6, Canada
Received for publication, July 25, 2002, and in revised form, October 28, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HOX homeodomain proteins bind short core
DNA sequences to control very specific developmental processes. DNA
binding affinity and sequence selectivity are increased by the
formation of cooperative complexes with the PBX homeodomain protein. A
conserved YPWM motif in the HOX protein is necessary for cooperative
binding with PBX. We have determined the structure of a PBX homeodomain
bound to a 14-mer DNA duplex. A relaxation-optimized procedure was
developed to measure DNA residual dipolar couplings at natural
abundance in the 20-kDa binary complex. When the PBX homeodomain
binds to DNA, a fourth The specification of segmental identity along the embryonic
anteroposterior axis is largely determined by Hox genes.
These genes encode transcription factors that bind DNA through a highly conserved 60-amino acid domain known as the homeodomain, which consists
of three The Hox gene cluster in Drosophila consists of
eight genes, the expression of which is directly related to their
location in the cluster. In mammals, four Hox clusters, A to
D, encompass a total of 39 genes. The mammalian HOX proteins are
classified into 13 paralog groups on the basis of their position in the
gene cluster and homology to the Drosophila Hox genes (2).
Paralogs are expressed in overlapping domains and possess both similar and unique functions. Despite their highly specific in vivo
activities, in vitro HOX homeodomain proteins bind to the
short DNA sequence TAAT with relatively low affinity (3). The formation
of cooperative DNA-binding complexes between HOX proteins and a
cofactor, PBX, increases both the affinity and specificity of HOX
proteins for DNA (4, 5).
PBX binds to the DNA sequence 5'-TGAT-3' (6, 7) through an atypical
three-amino acid loop extension
(TALE)1 homeodomain (8). The
extra amino acids, which extend the loop between the first and second
helices of the homeodomain (9), are necessary for cooperative DNA
binding with HOX proteins (10, 11), forming part of a hydrophobic
binding pocket for the YPWM motif (12, 13). The 15 amino acids
immediately C-terminal to the PBX homeodomain are highly conserved
among PBX (PBX1, -2, and -3) and related proteins
(Drosophila Exd, Caenorhabditis elegans ceh-20)
and increase the affinity of PBX for DNA and HOX proteins (11,
14).
The minimal elements required for formation of PBX-HOX complexes are
the PBX and HOX homeodomains and the HOX YPWM motif (15-19). The YPWM
motif is found at a variable distance (5-50 amino acids) N-terminal to
the homeodomain in paralog groups 1-8 of the mammalian Hox
cluster (16). Tryptophan residues that mediate cooperative binding are
also found in paralogs 9 and 10 (5, 20). Peptides that encompass the
HOX YPWM motif and flanking amino acids disrupt the formation of
PBX-HOX heterodimers (11, 16, 21) and also stimulate binding of the PBX
homeodomain to DNA. PBX-HOX heterodimers recognize the DNA sequence
5'-ATGATTNATNN-3' (5, 22). PBX binds to the TGAT
half-site, and HOX binds to the TNAT half-site. The
identities of the variable bases, N, are determined
by the HOX protein (5, 22, 23).
We have solved the solution structure of the extended PBX
homeodomain-DNA binary complex and studied the binding of a HOX-derived YPWM peptide to the complex. The structure provides insights into the
role of the C-terminal extension and the PBX-YPWM motif interaction in
increasing the affinity and specificity of HOX and PBX for DNA.
Sample Preparation--
Recombinant PBX protein, chemically
synthesized HOX-derived peptides, and DNA oligonucleotides (Fig. 1)
were purified as described previously (9). Plasmids encoding the HOXA1
and HOXD4 peptides were constructed by polymerase chain reaction
amplification using primers that incorporated stop codons and
BamHI and MfeI sites to clone HOXA1 amino acids
203-221 and HOXD4 amino acids 129-147 into pGEX-6P-1 (Amersham
Biosciences). The glutathione S-transferase fusion proteins were expressed in Escherichia coli BL21(DE3)
cells grown on M9 minimal media supplemented with
15N NH4Cl and U13C6
D-glucose as the sole nitrogen and carbon sources. The
proteins were purified according to the manufacturer's protocol (24), and the peptide was cleaved from glutathione S-transferase
with 50 units of Precission Protease (Amersham Biosciences) at 4 °C for 20 h. The resulting peptides were purified by reverse-phase chromatography on a C-18 column (Vydac), lyophilized, and resuspended in 20 mM sodium phosphate, pH 7.0, for NMR studies.
NMR Spectroscopy--
NMR experiments were recorded on Bruker
DRX 500, Varian INOVA 750, and Varian INOVA 800 spectrometers equipped
with pulsed-field gradient probes. Proton chemical shifts were
referenced to internal 2,2-dimethyl-2-silapentanesulfonic acid
at 0 ppm. 15N and 13C chemical shifts were
referenced to the proton spectrum using the ratio of gyromagnetic
moments ( Residual Dipolar Coupling (RDC)
Data--
1H-15N couplings were measured for
the PBX-DNA complex in isotropic solution and two aligned media:
10 mg/ml filamentous phage at 30 °C, and 5 mg/ml
q = 3.0 DMPC/DHPC bicelles at 37 °C. Initial values
for Da and R were estimated from the
RDC powder pattern (27). Final values of 13.5 and 0.6 in bicelles and
15.5 and 0.55 in phage for Da and R,
respectively, were determined using a variational method (28).
1H-13C residual couplings were measured by
taking twice the difference in peak position between a
t1-coupled 13C-HSQC and a
13C-HMQC. DNA RDCs were recorded in the presence and
absence of DMPC/DHPC bicelles in 100% D2O.
Structure Calculation--
100 structures of the PBX-DNA complex
were generated on the basis of the restraints listed in Table I.
The 20 lowest energy structures were chosen to represent
the solution structure of the binary complex. 1180 NOEs were assigned
manually, and eight iterations of ARIA (29) were used to obtain
additional assignments. The starting conformation for the calculation
consisted of the free PBX homeodomain (9) with the N-terminal arm and
C-terminal extension modeled as extended strands and placed next to
B-form DNA. Initially crystallography and NMR System (CNS; Yale
University) was used to generate 100 structures with the DNA duplex
frozen. The protein and DNA were then submitted to a second
round of dynamical annealing using Cartesian dynamics, starting from
the structures generated in the first round, with the dummy coordinate
system for the RDCs fixed and with NOE and RDC constraints for the DNA added.
The Extended PBX Homeodomain Binds to DNA, Forming a Fourth
A number of intermolecular NOEs are observed that position PBX
on the DNA duplex. The N-terminal arm lies in the minor groove of the
DNA. Many NOEs are observed between its arginine and lysine side chains
and the sugar protons of RDCs--
RDCs in the binary complex were measured in two
aligned media, lipid bicelles and filamentous phage. The
structure was refined using the RDCs measured in bicelle media. 60 1H-15N residual dipolar couplings were measured
for the backbone amides of the PBX homeodomain aligned in lipid
bicelles. The distribution of positive and negative RDCs reflects the
orientation of the four
28 aromatic and H1'-C1' residual dipolar couplings in the DNA were
measured at natural 13C abundance. Interference between
chemical shift anisotropy and dipolar relaxation mechanisms in the
binary complex resulted in a TROSY-like effect (30) in the
proton-coupled 13C-HSQC. The effect was most pronounced for
the aromatic and C1'-H1' cross-peaks, for which only the slowly
relaxing component of each 13C doublet was observed. The
large chemical shift anisotropy of aromatic carbons (>100 ppm) results
in favorable conditions for implementation of TROSY-type experiments in
the assignment of nucleic acids and aromatic residues of proteins
(31-33). The transverse relaxation rates of multiple quantum
coherences are longer than those of the corresponding single quantum
coherences. This phenomenon has also been exploited to improve
sensitivity in NMR experiments on large biomolecules (33-35). A
13C-HMQC spectrum was acquired to measure
1H-13C splittings. This method showed the
1H-decoupled chemical shifts and hence allowed calculation
of the 1H-13C couplings with only a single
component of the doublet (Fig. 3). The 13C-HMQC experiment
was more sensitive than an 1H-decoupled
13C-HSQC and yielded a more complete set of residual
dipolar couplings (data not shown). Residual dipolar couplings for both
the DNA and the PBX homeodomain were well satisfied in the structure
calculation (Fig. 3e).
HOXA1 and HOXD4-derived Peptides Are Structured in
Solution--
HOX-derived YPWM peptides were studied both free and
bound to the PBX-DNA complex. The peptides encompass the YPWM motif and flanking residues of HOXA1 and HOXD4 (Fig. 1). ROESY spectra were recorded at 15 °C and pH 5.0 for the free peptides. Medium range ROEs were observed between the aromatic ring of residue 4 and methionine 7 in both peptides (Fig. 4).
ROEs between the methyl groups of Val-9 and the aromatic rings of Phe-4
and Trp-6 are observed in HOXA1, and the side-chains of Val-2 and Tyr-4
of HOXD4 are close together. At a higher temperature and pH value
(30 °C, pH 6.8), the amide cross-peaks in the 15N-HSQC
are well dispersed, indicating that the structure is retained under
these conditions (Fig.
5a).
The HOXA1 Peptide Binds to a Preformed Hydrophobic
Pocket--
To discriminate between signals that arise from PBX
and HOXA1, samples were prepared in which either the peptide or the
homeodomain was 15N- or 15N- and
13C-labeled. Formation of the ternary complex was followed
by the recording of a series of 15N-HSQC spectra of the
labeled polypeptide during titration with the unlabeled component. The
amide cross-peaks for residues in PBX loop one and the C-terminal half
of helix three broaden and shift as HOXA1 is added to the PBX-DNA
complex. Residues in the C-terminal extension experience smaller
changes in chemical shift (9). Amide cross-peaks broaden, and
significant changes in chemical shift are observed for residues 4-10
of HOXA1 when the PBX-DNA complex is titrated into the peptide (Fig.
5). 1H-15N heteronuclear NOEs are positive for
amino acids 3-12, indicating that they are structured (Fig.
5c). In contrast to the binding of PBX to DNA, which is in
slow exchange on the NMR time scale, the HOXA1 peptide binds to the
PBX-DNA complex in an intermediate exchange regime. The low affinity of
a truncated HOXA1 peptide (residues 1-12 of the HOXA1 peptide) for the
PBX-DNA complex allowed the use of transferred NOEs to study its bound
conformation. A sample in which the peptide was in 20-fold molar excess
over the binary complex was prepared. Under these conditions
information about the bound state of the peptide is transferred to the
free state and observed. The proton resonances of the side chains of Phe-4, Trp-6, and Lys-8 were broadened compared with the free peptide
(Fig. 4). These side chains are involved in binding to PBX. More
intramolecular NOEs are observed in a transferred NOESY spectrum than
in the ROESY spectrum of the free HOXA1 peptide, indicating that it
becomes more structured on binding to the PBX-DNA complex.
Intermolecular NOEs between the aromatic rings of Phe-4 and Trp-6 and
the methyls of Leu-23a, and Met-7 and Ile-60 place the YPWM motif in a
pocket formed by loop one and helix three, as expected from the changes
in PBX chemical shifts.
15N and 13C-HSQC-NOESY spectra of PBX in the
binary and ternary complexes are very similar. Additional NOEs are
observed to the backbone amide of Ser-23b, which is
solvent-exposed in the binary complex. Some chemical shifts change for
the amino acids around the hydrophobic binding pocket, although the NOE
patterns are the same. The imino protons of base pairs 7 and 8 shift
slightly when HOXA1 is titrated into the PBX-DNA complex; otherwise the DNA spectrum is unchanged. The HOXA1 peptide binds to a preformed hydrophobic pocket bordered by the TALE and helices three and four of PBX.
Our determination of the binary PBX-DNA structure demonstrates
that the PBX homeodomain and the HOX YPWM motif interact via a "lock
and key" mechanism. On binding to DNA, the third The lengthening of the third The PBX-DNA structure was compared with the crystal structure of a
PBX-DNA-HOXB1 complex (12). The conformation of the PBX homeodomain is
very similar in the NMR and crystal structures (average
root-mean-square deviation 1.1 Å for the backbone atoms of the three
core ROESY spectra of the HOXA1 and HOXD4 peptides indicate that there is
some structure in the absence of PBX (Fig. 4a). Hydrophobic interactions between the aromatic ring of the tyrosine (or
phenylalanine) and the conserved methionine fold the peptide.
Interactions between conserved aromatic and hydrophobic residues
flanking the core YPWM motif also contribute. The HOXA1 peptide becomes
more structured on binding to the PBX homeodomain. Chemical shift
dispersion increases in 15N-HSQC spectra and more
intramolecular NOEs are observed. NMR studies at low temperature of two
six-amino acid peptides, TFDWMK and LFPWMR (i.e. residues
3-8 of our HOX peptides), have also demonstrated that the YPWM motif
is partially prefolded in solution (37). The conformation of the
PBX-bound HOXA1 peptide is similar to the corresponding region of HOXB1
in the crystal structure of the PBX-HOXB1 heterodimer (12). The HOX
YPWM motif binds to PBX through hydrophobic interactions. Contacts are
observed between the conserved tryptophan ring and the side chain of
Leu-23a in the extended loop in both the crystal structure and NOESY
spectra of the ternary complex. Intermolecular NOEs also place the
methionine and phenylalanine side chains within the hydrophobic binding
pocket as in the PBX-HOXB1 heterodimer. However, the relatively weak binding of the HOXA1 peptide to the PBX-DNA complex, and broad NMR line
widths indicate that the peptide experiences some conformational exchange that is not seen in the crystal structure. In the crystal, the
intact HOXB1 homeodomain and linker region stabilize the HOX YPWM
motif-PBX interaction.
The structures of the PBX-DNA and PBX-DNA-HOXB1 complexes show how
PBX-HOX heterodimers achieve increased DNA binding affinity and
different sequence selectivity (4, 5) and demonstrate the importance of
DNA-binding by PBX for heterodimer formation. The amino acid sequences
of the HOX N-terminal arm and the YPWM motif are the most important
determinants of binding site preference for PBX-HOX complexes (5, 23).
The insertion of the HOX YPWM "key" into the hydrophobic
"lock" on the surface of the PBX homeodomain tethers the HOX
homeodomain to the DNA duplex, resulting in the formation of more
stable contacts between the HOX N-terminal arm and DNA. Nevertheless,
the formation of PBX-HOX heterodimers is not the sole determinant of
the very precise in vivo functions of the different HOX
proteins. PBX-HOX binding sites have been identified in several gene
regulatory elements, including the Hoxb1 and
Hoxb2 r4 enhancers (38-42). Adjacent to these sites
are sequences recognized by PREP1 and MEIS homeodomain proteins that form trimeric complexes with HOX and PBX (42-46). Thus, the presence of a third partner may be expected to further modify protein-protein and subtle protein-DNA interactions in the DNA-bound homeodomains.
-helix is formed in the homeodomain. This
helix rigidifies the DNA recognition helix of PBX and forms a
hydrophobic binding site for the HOX YPWM peptide. The HOX
peptide itself shows some structure in solution and suggests that the
interaction between PBX and HOX is an example of "lock and key"
binding. The NMR structure explains the requirement of DNA for the
PBX-HOX interaction and the increased affinity of DNA binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices and an N-terminal arm. The third helix lies in the
major groove of the DNA and, along with the N-terminal arm, is
responsible for DNA recognition. Well conserved amino acid residues
contact DNA and form a hydrophobic core (1).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N/
H = 0.10132905,
C/
H = 0.25144952). Data were processed
using the Gifa program, versions 4.2 and 4.3 (25), and peak picking and
assignment were carried out with XEASY (26). Resonance assignments and
HNH
coupling constants for the PBX homeodomain and the HOXA1 peptide
were obtained by standard methods. The methyl groups of valine and
leucine side chains were assigned stereospecifically from a
13C-HSQC of a 10% 13C-labeled sample. DNA
assignments were made from a two-dimensional
1,
2-13C-filtered NOESY. Distance restraints for the
binary complex were derived from a 15N-edited HSQC-NOESY, a
13C-edited HSQC-NOESY, a two-dimensional
1,
2-13C-filtered NOESY, and a two-dimensional
2-13C-filtered NOESY. All NOESY spectra were recorded at
30 °C with mixing times of 150 ms. Intermolecular NOEs in the
ternary complex were obtained from a 15N-HSQC NOESY, a
13C-HSQC NOESY, and a two-dimensional transferred
NOE experiment recorded for a sample of 4.3 mM chemically
synthesized HOXA1 with 5% (mol:mol) PBX 1-78-DNA complex.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Helix--
We have determined the structure of the extended PBX
homeodomain (PBX 1-78) bound to a DNA duplex containing the PBX
recognition site 3'-ATGAT-5' (Fig. 1). An
ensemble of 20 structures, with a backbone root-mean-square deviation
of 0.54 Å (Table I), was chosen to
represent the structure of the complex in solution. The extended PBX
homeodomain consists of four
-helices and an N-terminal arm (Fig.
2). The first
-helix runs from Lys-10
to Phe-20, the second from Glu-28 to Ser-38, the third from Val-42 to
Lys-58, and the fourth from Ile-60 to Ala-72. The fourth
-helix forms on binding of the homeodomain to the DNA duplex and folds across
the homeodomain at an angle of approximately 65° to helix three,
crossing helix one.
View larger version (26K):
[in a new window]
Fig. 1.
Sequences of protein, DNA, and peptide
constructs. a, sequence of the PBX homeodomain. The
standard homeodomain numbering is shown above the sequence and the
numeric position in the full-length protein is shown below the
sequence. The extra three amino acids are labeled a,
b, and c, and the conserved residues of the
C-terminal extension are underlined. b, nucleic
acid sequence. The PBX recognition site is in bold. c,
HOX-derived peptides. The YPWM motif is indicated by bold type. The
numbering scheme used in this study is shown above the amino acid
sequences, and the position relative to the respective homeodomains is
shown below the sequences.
Structure calculations
View larger version (55K):
[in a new window]
Fig. 2.
The structure of the PBX-DNA complex.
Superposition of the heavy atoms of the 20 lowest energy structures of
the extended PBX homeodomain bound to a 14-mer DNA duplex. The backbone
of PBX residues 1-72 and the DNA duplex are illustrated. The N
terminus of the protein is labeled N; C indicates
the position of amino acid 72. Base pair 1 of the DNA is shown at the
bottom of the figure.
G4,
T5,
A6 and +A8, as well as the H2s
of
A6 and +A8. Particularly strong NOEs are observed between the
and
protons of Arg-5 and the H2 of
A6. Both Arg-5 and the TA base
pair (TNAT) are highly conserved in all homeodomains and recognition sites (1). The PBX three-amino acid
extension is accommodated in loop 1 in such a way that a conserved
Tyr-25-phosphate backbone contact is preserved. The third
-helix
sits in the major groove of the DNA. The methyl groups of isoleucine 54 in the third helix show strong NOEs to the sugars and bases of
A9,
A10, and
C11. Residual dipolar couplings were used in the
refinement of the structure to compensate for the lack of long range
NOEs in the nucleic acid.
-helices (Fig.
3). The PBX-DNA complex aligns with the
axis of the DNA duplex; the first three
-helices are roughly
perpendicular to the magnetic field, and the fourth
-helix is
parallel to the magnetic field. RDCs were also measured for PBX in the
ternary PBX-DNA-HOX complex aligned in filamentous phage (data not
shown). The orientation of both the binary and ternary complexes with respect to the external magnetic field was very similar.
View larger version (22K):
[in a new window]
Fig. 3.
1H-15N PBX
residual dipolar couplings. a, alignment of the PBX-DNA
complex with respect to the magnetic field in 5% DMPC/DHPC bicelles.
b, backbone amide residual dipolar couplings plotted against
residue number for the PBX homeodomain in bicelles, and c,
in filamentous phage. Maximum negative values for RDCs are observed
when the bond is parallel to the external magnetic field. Extreme
positive values are observed when the bond is perpendicular.
d, measurement of 1H-13C residual
dipolar couplings in DNA at natural abundance in lipid bicelles.
Superposition of the 13C-HMQC (blue) and
t1-coupled 13C-HSQC
(black) spectra for Ala-23 His-8 and Cys-14 His-6 in the
PBX-DNA complex. The upfield doublet component is barely observed for
Cys-14, one of the strongest resonances in the spectra. Experiments
were recorded in isotropic conditions (left) and aligned in
5% DMPC/DHPC bicelles (right) for 2.3 and 2.7 mM samples in 100% D2O at 500 MHz.
e, back correlation of DNA (blue) and PBX
(red) residual dipolar couplings in the binary complex.
Experimental and calculated values for the DNA
1H-13C couplings were normalized to
1H-15N values by a scale factor of 0.48, to
take into account the gyromagnetic ratios of 13C and
15N and the difference in CH and NH bond lengths.
View larger version (27K):
[in a new window]
Fig. 4.
The HOX YPWM motif is structured in
solution. a, ROESY spectrum of the free HOXA1 peptide.
b, transferred NOESY spectrum of 4.3 mM HOXA1
with 5% (mol:mol) PBX-DNA complex. Spectra were recorded at 500 MHz
and 15 °C. Medium and long range intramolecular ROEs/NOEs are
labeled. Schematic representations of medium and long range ROEs
(c) and trNOEs (d) in the free and complexed
peptide are shown below the two spectra.
View larger version (16K):
[in a new window]
Fig. 5.
Binding of the HOXA1 peptide to the PBX-DNA
complex. a, superposition of the 15N-HSQC
of the free (black) and bound (gray) HOXA1
peptide. Spectra recorded at 25 °C and 500 MHz. b,
schematic representation of chemical shift changes on binding to the
PBX-DNA complex. HN = ((
Hbound
Hfree × 5)2 + (
Nbound
Nfree)2). c, heteronuclear NOEs.
The ratio of the saturated versus unsaturated experiment
plotted by residue.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix of the
PBX homeodomain lengthens, and contact between Tyr-25 and the DNA
duplex brings the loop between the first two helices closer to the
third helix. This creates a hydrophobic pocket bordered by the TALE and
the C terminus of the third
-helix. The fourth
-helix, which
forms on binding to DNA, contributes an additional side to the pocket.
The HOX YPWM motif, which is partially folded by hydrophobic
interactions, inserts into this pocket. An increase in the number of
NOEs observed for the HOXA1 peptide indicates it becomes more
structured when it binds to PBX. No changes are observed in the
conformation of the PBX homeodomain on formation of the ternary complex.
-helix when PBX binds to DNA induces
the formation of a fourth
-helix in the C-terminal extension (9,
36). This
-helix folds over the homeodomain and contacts helix one,
thereby reducing the mobility of the third helix. This accounts for the
increased DNA-binding affinity of the extended PBX homeodomain, PBX
1-78, in comparison with the homeodomain, PBX 1-59 (11, 14, 15). No
additional DNA contacts are formed by the C-terminal extension. PBX
1-78 also demonstrates higher affinity for HOX proteins and peptides
(11, 14) because of stabilization of the hydrophobic binding pocket and
formation of additional PBX-HOX contacts in the C-terminal extension.
Conversely, HOX-derived peptides stimulate binding of the PBX
homeodomain to DNA (11, 16). The third
-helix is involved in both
DNA and YPWM binding interactions (9, 12, 13). The chemical shifts of
the imino protons of base pairs 7 and 8 change slightly when the HOXA1
peptide binds to the PBX-DNA complex. No direct contacts occur between
the peptide and the DNA, which suggests that the change in the
environment of the base pairs is a result of the repositioning of PBX
side chains. Proton resonances in the base and sugar moieties of +G7
and +A8 are among the broadest in the binary complex. The disordered
side chains of Arg-53 and Arg-55 may form more stable contacts with DNA
in the presence of the HOX YPWM motif.
-helices). Differences are seen in the positions of the
C-terminal half of helix three, helix four, and the extended loop. The
crystal structure includes the PBX homeodomain, the HOXB1 homeodomain
and YPWM motif, and a 20-base pair DNA duplex. Each homeodomain
introduces a slight bend in the DNA. In comparison, a single PBX
homeodomain is bound to a 14-mer DNA duplex in the NMR structure. Helix
three of the PBX homeodomain lies in the major groove of the DNA. In
the PBX-HOXB1 heterodimer, the N-terminal arm of the HOX homeodomain
contacts the same bases from the minor groove. The slightly different
conformation of the DNA results in the differences in the positions of
the third and fourth helices between the NMR and crystal structures.
The PBX-HOXB1 recognition site is 5'-TGATTGAT-3'. A single TGAT site
was used in the NMR structure determination to avoid homodimer
formation. In the NMR structure tyrosine 25 in the extended loop thus
contacts a guanine close to the end of the DNA duplex rather than the
thymine present in the crystal structure. Combined with the bend in the
DNA introduced by the HOXB1 homeodomain in the crystal structure, this
results in the shift in the position of the loop between helices one
and two. No changes were observed in the conformation of the PBX-DNA complex on binding of a HOXA1-derived YPWM peptide. The differences in
PBX in the NMR and crystal structures are a result of the sandwiching of the DNA between two homeodomains.
![]() |
ACKNOWLEDGEMENTS |
---|
750-MHz spectra were acquired at the Environmental Molecular Sciences Laboratory (a national scientific user facility sponsored by the United States Department of Energy Office of Biological and Environmental Research) located at Pacific Northwest National Laboratory, operated by Battelle for the Department of Energy. 800-MHz spectra were acquired at the National Highfield Nuclear Magnetic Resonance Centre facility located at the University of Alberta.
![]() |
FOOTNOTES |
---|
* This research was supported by Canadian Institutes of Health Research Grant MA14129 (to K. G.).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.
The atomic coordinates for the PBX-DNA complex (code 1LFU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Chemical shifts have been deposited in the BioMagnetic Resonance Bank (code 5349), University of Wisconsin, Madison, WI (www.bmrb.wisc.edu/).
§ Supported by a Canadian Institutes of Health doctoral award.
To whom correspondence should be addressed: Dept. of
Biochemistry, McIntyre Medical Sciences Bldg., McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.:
514-398-7287; Fax: 514-398-7384; E-mail:
kalle.gehring@mcgill.ca.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M207504200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TALE, three-amino acid loop extension; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; RDC, residual dipolar coupling; ROESY, rotating frame Overhauser enhancement spectroscopy; ROEs, rotating frame Overhauser enhancements; HSQC, heteronuclear single-quantum correlation; HMQC, heteronuclear multiple quantum coherence; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gehring, W. J., Affolter, M., and Bürglin, T. (1994) Annu. Rev. Biochem. 63, 487-526[CrossRef][Medline] [Order article via Infotrieve] |
2. | Krumlauf, R. (1994) Cell 78, 191-201[Medline] [Order article via Infotrieve] |
3. | Gehring, W. J., Qian, Y. Q., Billeter, M., Furukubo, T. K., Schier, A. F., Resendez, P. D., Affolter, M., Otting, G., and Wüthrich, K. (1994) Cell 78, 211-223[Medline] [Order article via Infotrieve] |
4. | Lu, Q., Knoepfler, P. S., Scheele, J., Wright, D. D., and Kamps, M. P. (1995) Mol. Cell. Biol. 15, 3786-3795[Abstract] |
5. | Chang, C. P., Brocchieri, L., Shen, W. F., Largman, C., and Cleary, M. L. (1996) Mol. Cell. Biol. 16, 1734-1745[Abstract] |
6. | Kamps, M. P., Murre, C., Sun, X. H., and Baltimore, D. (1990) Cell 60, 547-555[Medline] [Order article via Infotrieve] |
7. | Nourse, J., Mellentin, D., Galili, N., Walkinson, J., Stanbridge, E., Smith, S. D., and Cleary, M. L. (1990) Cell 60, 535-545[Medline] [Order article via Infotrieve] |
8. |
Bürglin, T. R.
(1997)
Nucleic Acids Res.
25,
4173-4180 |
9. | Sprules, T., Green, N., Featherstone, M., and Gehring, K. (2000) Biochemistry 39, 9943-9950[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Peltenburg, L. T.,
and Murre, C.
(1997)
Development
124,
1089-1098 |
11. | Lu, Q., and Kamps, M. P. (1996) Mol. Cell. Biol. 16, 1632-1640[Abstract] |
12. | Piper, D. E., Batchelor, A. H., Chang, C.-P., Cleary, M. L., and Wolberger, C. (1999) Cell 96, 587-597[Medline] [Order article via Infotrieve] |
13. | Passner, J. M., Ryoo, H. D., Shen, L., Mann, R. S., and Aggarwal, A. K. (1999) Nature 397, 714-719[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Green, N. C.,
Rambaldi, I.,
Teakles, J.,
and Featherstone, M. S.
(1998)
J. Biol. Chem.
273,
13273-13279 |
15. | Chang, C. P., Shen, W. F., Rozenfeld, S., Lawrence, H. J., Largman, C., and Cleary, M. L. (1995) Genes Dev. 9, 663-674[Abstract] |
16. | Knoepfler, P. S., and Kamps, M. P. (1995) Mol. Cell. Biol. 15, 5811-5819[Abstract] |
17. | Mann, R. S., and Chan, S. K. (1996) Trends Genet. 12, 258-262[CrossRef][Medline] [Order article via Infotrieve] |
18. | Neuteboom, S. T., Peltenburg, L. T., van Dijk, M. A., and Murre, C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9166-9170[Abstract] |
19. | Phelan, M. L., Rambaldi, I., and Featherstone, M. S. (1995) Mol. Cell. Biol. 15, 3989-3997[Abstract] |
20. |
Shen, W. F.,
Rozenfeld, S.,
Lawrence, H. J.,
and Largman, C.
(1997)
J. Biol. Chem.
272,
8198-8206 |
21. |
Shanmugam, K.,
Featherstone, M. S.,
and Saragovi, H. U.
(1997)
J. Biol. Chem.
272,
19081-19087 |
22. |
Chan, S. K.,
and Mann, R. S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5223-5228 |
23. |
Phelan, M. L.,
and Featherstone, M. S.
(1997)
J. Biol. Chem.
272,
8635-8643 |
24. | Amersham Biosciences. (1998) Glutathione Sepharose 4B , Amersham Biosciences |
25. | Pons, J. L., Malliavin, T. E., and Delsuc, M. A. (1996) J. Biomol. NMR 8, 445-452 |
26. | Bartels, C., Xia, T.-H., Billeter, M., Guntert, P., and Wüthrich, K. (1995) J. Biomol. NMR 5, 1-10[Medline] [Order article via Infotrieve] |
27. | Clore, G. M., Gronenborn, A. M., and Bax, A. (1998) J. Magn. Reson. 133, 216-221[CrossRef][Medline] [Order article via Infotrieve] |
28. | Clore, G. M., Gronenborn, A. M., and Tjandra, N. (1998) J. Magn. Reson. 131, 159-162[CrossRef][Medline] [Order article via Infotrieve] |
29. | Nilges, M., Macias, M. J., O'Donoghue, S. I., and Oschkinat, H. (1997) J. Mol. Biol. 269, 408-422[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Pervushin, K.,
Riek, R.,
Wider, G.,
and Wüthrich, K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12366-12371 |
31. | Pervushin, K., Riek, R., Wider, G., and Wüthrich, K. (1998) J. Am. Chem. Soc. 120, 6394-6400[CrossRef] |
32. | Riek, R., Pervushin, K., Fernandez, C., Kainosho, M., and Wüthrich, K. (2001) J. Am. Chem. Soc. 123, 658-664[CrossRef][Medline] [Order article via Infotrieve] |
33. | Fiala, R., Czernek, J., and Sklenar, V. (2000) J. Biomol. NMR 16, 291-302[CrossRef][Medline] [Order article via Infotrieve] |
34. | Kuboniwa, H., Grzesiek, S., Delaglio, F., and Bax, A. (1994) J. Biomol. NMR 4, 871-878[Medline] [Order article via Infotrieve] |
35. | Marino, J. P., Diener, J. L., Moore, P. B., and Griesinger, C. (1997) J. Am. Chem. Soc. 119, 7361-7366[CrossRef] |
36. | Jabet, C., Gitti, R., Summers, M. F., and Wolberger, C. (1999) J. Mol. Biol. 291, 521-530[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Slupsky, C. M.,
Sykes, D. B.,
Gay, G. L.,
and Sykes, B. D.
(2001)
Protein Sci.
10,
1244-1253 |
38. |
Chen, J.,
and Ruley, H. E.
(1998)
J. Biol. Chem.
273,
24670-24675 |
39. |
Pan, L.,
Xie, Y.,
Black, T. A.,
Jones, C. A.,
Pruitt, S. C.,
and Gross, K. W.
(2001)
J. Biol. Chem.
276,
32489-32494 |
40. |
Ferretti, E.,
Marshall, H.,
Popperl, H.,
Maconochie, M.,
Krumlauf, R.,
and Blasi, F.
(2000)
Development
127,
155-166 |
41. | Maconochie, M. K., Nonchev, S., Studer, M., Chan, S. K., Pöpperl, H., Sham, M. H., Mann, R. S., and Krumlauf, R. (1997) Genes Dev. 11, 1885-1895[Abstract] |
42. |
Jacobs, Y.,
Schnabel, C. A.,
and Cleary, M. L.
(1999)
Mol. Cell. Biol.
19,
5134-5142 |
43. | Chang, C. P., Jacobs, Y., Nakamura, T., Jenkins, N. A., Copeland, N. G., and Cleary, M. L. (1997) Mol. Cell. Biol. 17, 5679-5687[Abstract] |
44. |
Berthelsen, J.,
Zappavigna, V.,
Ferretti, E.,
Mavilio, F.,
and Blasi, F.
(1998)
EMBO J.
17,
1434-1445 |
45. | Ferretti, E., Schulz, H., Talarico, D., Blasi, F., and Berthelsen, J. (1999) Mech. Dev. 83, 53-64[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Shanmugam, K.,
Green, N. C.,
Rambaldi, I.,
Saragovi, H. U.,
and Featherstone, M. S.
(1999)
Mol. Cell. Biol.
19,
7577-7588 |