From the Department of Biochemistry, The
Hong Kong University of Science and Technology, Clear Water Bay,
Kowloon, Hong Kong, People's Republic of China and the
§ Howard Hughes Medical Institute and Department of
Neurobiology, Massachusetts General Hospital and Harvard Medical
School, Boston, Massachusetts 02114
Received for publication, November 14, 2000, and in revised form, January 8, 2001
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ABSTRACT |
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Cytoplasmic dynein is a large, multisubunit
molecular motor that translocates cargoes toward the minus ends of
microtubules. Proper functioning of the dynein motor requires precise
assembly of its various subunits. Using purified recombinant proteins, we show that the highly conserved 8-kDa light chain (DLC8) binds to the
intermediate chain of the dynein complex. The DLC8-binding region was
mapped to a highly conserved 10-residue fragment (amino acid sequence
SYSKETQTPL) C-terminal to the second alternative splicing site of
dynein intermediate chain. Yeast two-hybrid screening using DLC8
as bait identified numerous additional DLC8-binding proteins.
Biochemical and mutational analysis of selected DLC8-binding proteins
revealed that DLC8 binds to a consensus sequence containing a
(K/R)XTQT motif. The (K/R)XTQT motif interacts
with the common target-accepting grooves of DLC8 dimer. The role of
each conserved amino acid residue in this pentapeptide motif in
supporting complex formation with DLC8 was systematically studied using
site-directed mutagenesis.
Cytoplasmic dynein is a microtubule-based molecular motor that has
been implicated in a wide variety of functions including retrograde
organelle movement, nuclear migration, mitotic spindle alignment, and
axonal transport (1-3). The enzyme is a multisubunit complex assembly
containing two molecules of heavy chains
(DHC1, ~530 kDa), several
intermediate chains (DIC, ~74 kDa) and light intermediate chains
(DLIC, 53-59 kDa), and a number of light chains (DLC, 8-22 kDa). The
heavy chains of dynein contain ATPase activity essential for force
generation and are also responsible for attaching the motor complex to
microtubules. The divergent N-terminal one-third of DHC contains the
DIC-binding site. DICs may function as linkers of cargoes to the dynein
motor through direct binding to dynactin (4). The functions of other
dynein polypeptides are largely unknown.
The 8-kDa light chain (DLC8) of cytoplasmic dynein was originally
identified as a light chain of Chlamydomonas outer arm
axonemal dynein (5-7). The protein was subsequently shown to be a
stoichiometric component of cytoplasmic dynein and of actin-based motor
myosin V (8). Mutational studies showed that DLC8 is required for proper functioning of cytoplasmic dynein including retrograde intraflagellar transport (9), nuclear migration, and motor complex
localization (10). However, it is not known how DLC8 is assembled into
the cytoplasmic dynein motor complex, although it was suggested that
the protein is associated with DIC in the axonemal dynein complex
(6).
DLC8 contains 89 amino acid residues and is highly conserved throughout
evolution (>90% amino acid sequence identity from Caenorhabditis elegans to humans). Other than binding to
certain subunits of motor proteins, DLC8 has also been shown to
interact with several cellular targets with diverse functions. DLC8
binds to neuronal nitric-oxide synthase (11, 12), proapoptotic Bcl-2 family protein Bim (13), Drosophilia mRNA
localization protein Swallow (14), transcriptional regulator I We report here that DLC8 binds to a conserved
(K/R)XTQT amino acid sequence motif in a wide variety
of protein partners. Such a motif is present in DIC of cytoplasmic
dynein and mediates association of DLC8 and DIC in the dynein complex.
Yeast Two-hybrid Screen of DLC8-binding Proteins--
Yeast-two
hybrid screening was performed as described previously (16). Briefly,
DLC8 was subcloned into pBHA (LexA fusion vector) and used to
separately screen ~1 × 106 clones each from human
and rat cDNA libraries constructed in pGAD10 (GAL4 activation
vector; CLONTECH).
Expression Constructs--
The full-length mouse DIC gene was
constructed by assembling three partially overlapping genomic clones
using standard PCR and DNA ligation methods. The DIC fragments were
generous gifts from Dr. L. C. Tsui (20). The full-length DIC
coding DNA fragment was inserted into the
EcoRI/BamHI sites of an in-house-modified pET32a
plasmid vector (Novagen). Various truncation and point mutants of DICs
were cloned into pGEX-4T-1 vector (Amersham Pharmacia Biotech) using
standard recombinant DNA methods. The plasmids harboring the
full-length DIC gene and various mutant DIC genes were transformed into
Escherichia coli BL21(DE3) host cells for fusion protein production.
The C-terminal fragment of c82 (gene product of clone 82 identified
from a yeast two-hybrid screening for DLC8-binding proteins; see Table
I) containing amino acid residues 119-199 of the native protein was
amplified by PCR using the coding strand primer
5'-CCGGAATTCGAGAGATTGCAGGGTCTG-3' and noncoding strand primer
5'-CCGCTCGAGCTAGAGGCTGGGTCTAC-3'. The PCR-amplified c82 fragment was
inserted into the EcoRI/XhoI sites of pGEX-4T-1.
Various GST-fused c82 truncation mutants used in this study were
generated using standard PCR techniques. The GST-fused BS69 fragment
was prepared using a method similar to that described for c82, except
that two primers specific for BS69 were used in the PCR amplification.
Bacterial expression vectors of GST-fused BimL (long form)
and BimS (short form), both lacking the hydrophobic
transmembrane region and the BH3 domain, were constructed by PCR using
full-length BimL and BimS (gifts from Dr. A. Strasser) as templates, respectively, using a pair of primers:
5'-CGGGGATCCATGGCCAAGCAAC-3' (sense), and
5'-CCGCTCGAGTCACTCCTGTGCGATCC-3' (antisense).
Expression and Purification of the Fusion Proteins--
To
express the full-length DIC and its truncated mutants, host cells
transformed with the DIC expression plasmids were grown in LB medium at
37 °C until A600 reached ~1.2. The
expression of the proteins was induced by the addition of
isopropyl-1-thio-
Various GST-fused DIC (as well as c82 and BS69) truncated and point
mutants were expressed in soluble forms and purified using GSH-Sepharose affinity columns (Amersham Pharmacia Biotech) following the instructions of the manufacturer. The purified GST fusion proteins
were dialyzed against 1× phosphate-buffered saline buffer (pH 7.4) to
remove GSH, and the protein samples were directly used for DLC8 binding
assay experiments. To express GST·BimL and GST·BimS, host cells containing the Bim plasmid
constructs were grown in LB medium at 37 °C, reaching an
A600 of ~1.0. The temperature of the bacterial
cultures was then lowered to 25 °C before induction of GST·Bim
expression. Purification of GST·Bim proteins followed the procedure
described for the purification of the GST·DIC mutant proteins.
Preparation of pure and untagged DLC8 was described in our earlier work
(12).
Pull-down and Peptide Competition Experiments--
Direct
interactions between DLC8 and various GST-fused proteins were assayed
in phosphate-buffered saline buffer (pH 7.4). Equal molar amounts of
DLC8 and one of the GST fusion proteins (0.6 nmol each) were mixed in
100 µl of the assay buffer. The GST fusion protein-DLC8 complexes
were pelleted by 30 µl of fresh GSH-Sepharose beads. The pellets were
washed three times with 0.5 ml of the assay buffer and subsequently
boiled with 30 µl of 2× SDS-PAGE sample buffer. The intensity of the
DLC8 band on SDS-PAGE gels was used to judge the strength of the
interaction between DLC8 and various GST fusion proteins.
An 11-residue synthetic peptide (VSYSKETQTPL), corresponding to amino
acid residues 147-157 of DIC, was commercially synthesized. To assay
competition between the synthetic peptide and DIC for DLC8, increasing
amounts of the peptide (from 0 to 200 molar ratio amounts of DIC) were
included in the GST·DIC/DLC8 mixture. Residual GST·DIC-bound DLC8
was assayed using GSH-Sepharose affinity "pull-down" followed by
SDS-PAGE as described above.
NMR Experiments--
1H,15N HSQC
spectra of 15N-labeled DLC8 complexed with various
synthetic peptides were acquired on a Varian Inova 500 MHz spectrometer equipped with a z-gradient shielded triple resonance probe.
All NMR spectra were recorded at 30 °C, with a protein concentration of ~0.5 mM dissolved in 100 mM potassium
phosphate buffer, pH 7.0.
DLC8 Binds to an 11-Residue Fragment Located within a Highly
Conserved Region of the N-terminal Domain of DIC--
To understand
how DLC8 is assembled in cytoplasmic dynein, we set out to identify the
binding partner of DLC8 within the dynein complex. Given the overall
similarity between axonemal and cytoplasmic dynein complexes, we
suspected that DLC8 may also bind to DIC in cytoplasmic dynein. The
gene encoding full-length DIC was assembled from three overlapping
genomic clones (corresponding to the long form of DIC,
GenBankTM accession number AF063229). His-tagged
full-length DIC was expressed in E. coli cells and purified
in its denatured form. DIC was successfully refolded, and the refolded
DIC was assayed for its binding to DLC8. Affinity pull-down
assay indicates that the full-length DIC binds directly to DLC8 (Fig.
1B). The circular dichroism
spectrum of the refolded, full-length DIC indicated that the protein
has a well folded structure in solution (data not shown).
To further define the DLC8-binding region, we created a series of
truncation mutants of DIC as GST fusion proteins (Fig. 1A). The purified proteins were subsequently assayed for binding to DLC8 by
simple mixing and GSH-Sepharose bead pull-down. The DLC8-binding region
was mapped within the N-terminal domain of DIC (Fig. 1C). An
11-residue fragment corresponding to amino acid residues 147-157 (VSYSKETQTPL) of DIC was sufficient to bind to DLC8 (Fig. 1C, lane 6).
We verified the interaction between DLC8 and the 11-residue peptide
fragment of DIC derived from the truncation mapping experiment using
two different approaches. In the first experiment, we titrated 15N-labeled DLC8 with a synthetic peptide corresponding to
residues 147-157 of DIC (the DIC peptide). Fig.
2, A and B show the
1H,15N HSQC spectra of DLC8 before and after
binding to the DIC peptide. The chemical shift changes of DLC8
resulting from the DIC peptide binding are summarized using the minimal
shift perturbation approach (Fig. 2C). These data show that
the DIC peptide specifically binds to DLC8 and that the peptide-binding
region is located at the target-binding groove formed by the The 11-Residue Peptide Fragment Is the Only DLC8-binding Domain of
DIC--
The results presented in Figs. 1 and 2 demonstrate that the
11-residue peptide fragment (residues 147-157) of DIC is sufficient to
bind to DLC8. Is this 11-residue fragment in DIC the only binding site
for DLC8? To address this question, we disrupted the 11-residue DLC8-binding site by deleting residues 151-155 (KETQT) of DIC. This
deletion mutant did not bind to DLC8, indicating that the 11-residue
peptide fragment mapped in this study is the only DLC8-binding site in
DIC (Fig. 3).
(K/R)XTQT Is a Common DLC8 Recognition Motif--
To identify
additional proteins that interact with DLC8, we performed a yeast
two-hybrid screen using DLC8 as bait with both human and mouse brain
cDNA libraries. A large number of specific interactors were
isolated in the screen (see a partial list in Table
I), many of which represent previously
unidentified, potential DLC8-binding partners. We focus on a subset of
the genes in Table I to study their interaction with DLC8.
The first gene we chose was clone 82 (c82), because the full-length
sequence of c82 was recently deposited in GenBankTM. The
fragment isolated from the two-hybrid screen encodes the C-terminal
half of the c82 protein (residues 119-199). The interaction between
c82 and DLC8 was confirmed in vitro using purified
recombinant proteins (Fig. 4). Using a
series of deletion mutants, the DLC8-binding domain of c82 was mapped
to a 14-residue peptide fragment with amino acid sequence
159VGMHSKGTQTAKEE172 (Fig. 4).
The cDNA fragment of BS69 isolated from the DLC8 two-hybrid screen
encodes amino acid residues 396-424 of the protein. GST-fused BS69-(396-424) robustly bound to DLC8 in vitro,
indicating that the DLC8-binding region in BS69 is contained within
this 29-amino acid segment (Fig.
5A). A stretch of amino acids
similar to the DLC8-binding regions of DIC and c82 was noted within
this 29-residue fragment, as was
410MLHRSTQTTN419.
The proapoptotic protein Bim was previously shown to interact
specifically with DLC8, and the DLC8-binding domain was mapped to a
region of ~30 amino acid residues that were absent in the short form
(BimS) of the alternatively spliced gene product (13). We
confirmed this interaction between BimL and DLC8 in
vitro using purified recombinant proteins (Fig.
6A). Additionally, we
synthesized three overlapping peptides to map the minimal DLC8-binding
domain of BimL (Fig. 6B). NMR titration of
15N-labeled protein showed that all three peptides bound to
the protein in an essentially identical manner (data not shown). The shortest peptide used in this experiment contains only nine amino acid
residues (MSCDKSTQT). The 1H,15N
HSQC spectrum of this 9-residue peptide-bound form of DLC8 is shown in
Fig. 6B; the data suggest that this 9-residue peptide forms
a stable complex with DLC8 (18).
We aligned the amino acid sequences of the DLC8-binding domains of
various targets mapped in this study (Fig.
7). In this alignment, we also included
putative DLC8-binding domains of Swallow and rabies virus P protein,
two recently identified DLC8-binding proteins (14, 21, 22). The
(K/R)XTQT motif was present in each of these DLC8
interactors and probably represents the consensus DLC8-binding sequence
of these proteins (Fig. 7).
Mutational Analysis of the Consensus DLC8-binding Motif--
To
gain more insights into the interaction between DLC8 and the
(K/R)XTQT motif, we mutated each consensus amino acid in the motif and assayed for the effect. To facilitate the binding assay, we
fused the DLC8-binding domain of DIC with GST. In GST pull-down assays,
mutation of the Gln residue in the (K/R)XTQT motif to either
Ala or Gly completely abolished binding of the peptide to DLC8 (Fig.
8B). Mutation of either Thr
residue in the motif to Gly also abolished interaction between the
peptide and DLC8. Mutation of the first Thr (N-terminal to the Gln) to
Ala greatly inhibited the binding, suggesting that the hydroxyl group
of the Thr plays an active role in the complex formation. Mutation of the second Thr to Ser also significantly weakened the interaction, pointing to a possible role of the methyl group in the Thr side chain
in peptide-DLC8 complex formation. Mutations of the Lys residue to a
neutral Ala or a negatively charged Glu weakened the binding of the
peptide to DLC8. However, the magnitude of the inhibition by the Lys
mutations was lower than those observed with other point mutations.
Taken together, the data in Fig. 8 indicate that all four consensus
amino acid residues in the (K/R)XTQT motif play active
roles in supporting productive complex formation between DLC8 and its
target peptides. The data also indicate that the Thr-Gln-Thr tripeptide
is likely to play a more dominant role in the peptide-DLC8
interaction.
As a multicomponent macromolecular complex, precise organization
of various subunits of the dynein complex is essential for the motor to
function properly. Recent molecular analysis of the cytoplasmic dynein
complex has provided a detailed picture of the assembly of the DHC,
DIC, and DLIC subunits in the motor complex (23, 24). However, little
is known regarding the assembly of the light chains. Both cytoplasmic
and axonemal dyneins share the highly conserved DLC8. In
Chlamydomonas outer arm dynein, DLC8 was suggested to
associate with the intermediate chains of the motor complex (7). By
analogy, it was suggested that DLC8 is also assembled into the
cytoplasmic dynein by binding to DIC subunits of the motor complex (8),
although the flagellar and cytoplasmic DICs share limited sequence
identity (particularly in the N-terminal half of the proteins). Using
purified recombinant proteins, we show here that DLC8 can indeed bind
to the full-length intermediate chain of cytoplasmic dynein (Fig. 1).
An 11-residue fragment in the N-terminal half of DIC is necessary and
sufficient for binding to DLC8. Our data is consistent with an earlier
observation that DLC8 and DIC exist at a 1:1 stoichiometric ratio in
cytoplasmic dynein (8).
Mammalian DIC is encoded by two different genes, and each gene
generates multiple protein products by alternative splicing (4, 26).
The 11-residue DLC8-binding domain of mouse DIC mapped in this study
contains the amino acid sequence
148VSYSKETQTPL157, and this fragment is located
C-terminal to the second alternative splicing site of DIC. Within this
11-residue fragment, the 5-residue KETQT motif is likely to be
responsible for the interaction of DIC to DLC8 (Figs. 6 and 8; see also
Ref. 18). This 5-residue KETQT motif is present in all splice
isoforms of both DIC gene products. Furthermore, the KETQT motif is
highly conserved in cytoplasmic DICs throughout evolution (27). Given
the fundamental roles that DLC8 plays in dynein motor function (9, 10),
it might be expected that the DLC8-binding site should always remain available in various forms of DIC (and hence the dynein complex). We
further note that the DLC8-binding domain does not overlap with the
dynactin- and Tctex-1-binding sites of DIC, because dynactin binds to
the first 120-amino acid residue fragment of DIC (4, 28). If DLC8
indeed acts as a dynein cargo adaptor, a single dynein complex may thus
interact simultaneously with multiple cargoes.
The amino acid sequence of DLC8 is highly conserved throughout
evolution, and the protein is ubiquitously expressed in various tissues. As such, DLC8 was proposed to be a multifunctional regulatory protein, in addition to functioning as a light chain of the dynein complex (11, 12, 25). To gain more insights into the range of proteins
that interact with DLC8, we performed a yeast two-hybrid screen using
DLC8 as bait. No obvious amino acid sequence homology or functional
relationship can be identified among the proteins listed in Table I or
among other proteins previously identified as binding to DLC8. Given
that DLC8 can bind to short peptide fragments of ~10 amino acid
residues (12, 17, 18), we performed detailed mapping analysis of
DLC8-binding domains in several additional DLC8-binding proteins either
identified from our yeast two-hybrid screen (c82 and BS69) or
previously discovered (Bim). The DLC8-binding domains of these proteins
are contained within short stretches of amino acids (9-29 amino acid
residues). Sequence alignment analysis of the DLC8-binding domains
immediately suggests that the 5-residue (K/R)XTQT sequence
is a common DLC8 recognition motif in target proteins (Fig. 7). We also
predict that DLC8 is likely to bind to Swallow via the
291KATQT295 sequence immediately C-terminal to
the coiled-coil domain of Swallow. Our prediction is consistent with
experimental data showing that the DLC8-binding domain lies within
amino acid residues 197-295 of Swallow (14). Recently, DLC8 was shown
to interact with rabies virus P protein (the It is important to point out that the (K/R)XTQT motif is not
the only DLC8 recognition sequence in target proteins. For example, the
amino acid sequence of the DLC8-binding domain of nNOS is clearly
different from the (K/R)XTQT motif (Fig. 7). We were not able to find a (K/R)XTQT motif in a number of other known
DLC8-binding proteins including myosin V, GKAP, AIBC1, and
KIAA0710. We showed earlier that DLC8 binds to a (K/R)XTQT
motif-containing peptide and a peptide encompassing the DLC8-binding
domain of nNOS with remarkably similar mechanisms, although the two
peptides share little amino acid sequence identity (18). We have also
shown that the structural and dynamic properties of the target-binding site of DLC8 is uniquely suited to bind to peptide fragments
with diverse amino acid sequences without sacrificing target
binding specificity and
affinity.2 Therefore, it is
perhaps not surprising that DLC8 can bind a number of other target
proteins, even though these proteins do not contain a
(K/R)XTQT motif. We would also like to emphasize that the
DLC8-binding proteins listed in Table I are only potential DLC8
targets, because we only used partial fragments of the proteins for
binding studies. Further in vitro and in vivo
studies are required to substantiate whether these proteins are genuine
DLC8-binding targets under physiological conditions.
Our earlier structural studies showed that the (K/R)XTQT
motif forms a In summary, we have shown that DLC8 binds to the N-terminal domain of
cytoplasmic DIC via a highly conserved (K/R)XTQT motif. The
data firmly establish that DLC8 is assembled into the cytoplasmic dynein by binding to its intermediate chain located at the base of the
motor complex. In addition, we further show that the
(K/R)XTQT motif represents a general DLC8 recognition
sequence in its diverse target proteins. The experimental data
presented in this work, together with our earlier structural studies,
reinforce the hypothesis that DLC8 probably acts as a multifunctional
regulatory protein by binding to a large number of functionally
unrelated proteins.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B
(15), and postsynaptic scaffold protein GKAP (16). The
DLC8-binding domain of nNOS was mapped to a short stretch of
amino acid residues (12, 17). The other known DLC8-binding proteins
neither contain homologous DLC8-interacting sequences found in nNOS nor
bear obvious amino acid sequence similarities among each other.
Elucidation of the molecular basis of the interactions between DLC8 and
its diverse target proteins represents an important step in
understanding the function of this versatile protein. Structural
studies showed that DLC8 contains two identical target-binding grooves
located at opposite faces of the protein dimer interface (18). DLC8 is
capable of binding to short peptide fragments of ~10 amino acid
residues from its targets. The target peptides bind to DLC8 in an
antiparallel
-strand structure by pairing with the
-strand located at the base of each target-accepting groove (17, 18).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside to a final
concentration of ~0.5 mM. Protein expression continued for ~3 h at 37 °C, and the cells were then harvested by
centrifugation and stored at
80 °C prior to protein purification.
The full-length DIC containing a hexahistidine tag at the N terminus
was expressed in inclusion bodies. The pelleted bacterial cells were
resuspended in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml antipain prior to cell lysis
using sonication. The inclusion bodies were then washed extensively
using 50 mM Tris-HCl buffer (pH 7.5) containing 1 M urea and 0.5% Triton X-100. The washed inclusion bodies
were solubilized in 50 mM Tris-HCl buffer (pH 7.9)
containing 5 mM imidazole, 0.5 M NaCl, and 6 M guanidine HCl at room temperature. The denatured
protein was then passed through a
Ni2+-nitrilotriacetic acid affinity column following
the procedure described by the manufacturer (Novagen) for proteins
under denaturing conditions. Refolding of full-length DIC was achieved
by a single step dialysis of the denatured protein against 1×
phosphate-buffered saline buffer (pH 7.4). The refolded protein was
recovered from the supernatant after centrifugation.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mapping of the DLC8-binding site in DIC.
A, schematic diagram of full-length DIC and various DIC
fragments used for assaying binding to DLC8. To facilitate refolding,
the full-length DIC was expressed as a His-tagged protein. All of the
DIC fragments were fused to GST. The binding of various forms of DIC
with DLC8 is also summarized in the figure. B and
C, Coomassie Blue staining of SDS-PAGE gels showing the
interactions between various purified DIC fragments and DLC8. For the
full-length (FL) DIC,
Ni2+-nitrilotriacetic acid
(Ni2+-NTA) beads were used as a
negative control in the binding experiment (B). The
Ni2+-nitrilotriacetic acid beads contain some nonspecific
binding to DLC8. To avoid possible confusion, the rest of the DIC
fragments were prepared as GST fusion proteins. Purified GST was used
as the negative control for the binding interactions between DIC
fragments and DLC8. The lane numbers in C correspond to the
construct numbers in A. MW, molecular weight
markers.
2,
3
strands, the loop linking
2 and
3, and part of the
2 helix
(see "Discussion" for details). The observation of slow chemical
exchange between the apo-DLC8 and peptide-bound DLC8 during titration
indicates that the DIC peptide binds to DLC8 with a high affinity (data
not shown). NMR titration experiments also showed that the DIC peptide
binds to DLC8 with a 1:1 stoichiometry. In the second approach, we
performed a binding competition experiment using the DIC peptide.
Increasing molar amounts of the DIC peptide specifically competed with
DIC protein for binding to DLC8 (Fig. 2D). As a negative
control, an 11-residue peptide containing a partial Tctex-1-binding
domain of DIC (amino acid sequence LGRRLHKLGVS) was found not to
compete with DIC for binding to DLC8 (data not shown and Ref. 28).
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Fig. 2.
The DIC peptide specifically binds to
DLC8. 1H,15N HSQC spectra of
15N-labeled free DLC8 (A) and the protein
complexed with the DIC peptide (B). The assignments of
selected amino acid residues of both free DLC8 and its complex with the
DIC peptides are labeled with amino acid residue numbers and names. The
large chemical shift changes of DLC8 resulting from DIC peptide binding
indicate that DLC8 undergoes a significant conformational change upon
binding to the DIC peptide. C, chemical shift changes of
DLC8 resulting from the binding of the DIC peptide expressed using the
minimal shift perturbation approach (19). The combined 1H
and 15N chemical shift changes are defined according to
Equation 1.
The scaling factor (
(Eq. 1)
N) used to normalize the
1H and 15N chemical shifts is 0.17. The
secondary structure of DLC8 is also included in the figure.
D, the DIC peptide competes with DIC for DLC8 in a
dose-dependent manner. In this experiment, equimolar
amounts of GST·DIC and DLC8 were mixed with increasing amounts of the
DIC peptide. The remaining DLC8 complexed with GST·DIC was pulled
down by GSH-Sepharose affinity beads and analyzed by SDS-PAGE followed
by Coomassie Blue staining. MW, molecular weight
markers.
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Fig. 3.
Deletion of the KETQT pentapeptide
sequence from DIC disrupts its binding to DLC8. Equivalent amounts
of purified GST·DIC and GST·DIC( KETQT) were used in the binding
assay following the procedure described in the legend to Fig.
1C. Pure GST was used as a negative control in the
experiment. The SDS-PAGE gel was stained with Coomassie Blue.
DLC8-binding proteins identified through yeast two-hybrid screening
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Fig. 4.
Mapping the DLC8-binding domain of c82.
A, schematic diagram showing the GST·c82 fragments used in
the experiment. The amino acid sequence of the smallest DLC8-binding
fragment is also shown in the figure. B, Coomassie Blue
staining of the SDS-PAGE gel showing the interactions between various
purified c82 fragments and DLC8. MW, molecular weight
markers.
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Fig. 5.
Interaction of the BS69 fragment with
DLC8. A, Coomassie Blue staining of the SDS-PAGE gel
showing the interactions between DLC8 and a 29-residue fragment
of BS69 fused with GST. This 29-residue fragment was identified six
times in the yeast two-hybrid screening using DLC8 as bait.
B, a partial amino acid sequence showing the potential
DLC8-binding region of BS69.
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Fig. 6.
Interaction between Bim and DLC8.
A, Coomassie Blue staining of the SDS-PAGE gel showing the
interaction between alternatively spliced forms of Bim
(BimL and BimS) with DLC8. MW,
molecular weight makers. B, amino acid sequences of the
three overlapping synthetic peptides used to map the minimal
DLC8-binding domain of Bim. To study the interaction between the
peptides and DLC8, we titrated 15N-labeled DLC8 with each
peptide to observe chemical shift changes of the
1H,15N HSQC spectra of the protein.
C, 1H,15N HSQC spectrum of
15N-labeled DLC8 complexed with the 9-residue Bim peptide
shown in B. The assignment of the protein in the complex was
obtained from our earlier work (18).
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Fig. 7.
Sequence alignment of the DLC8-binding
domains from its various targets. The DLC8-binding domains mapped
in this study are aligned with each other. The putative DLC8-binding
domains of Swallow and rabies virus P protein (P-PV)
are also included in this alignment. The consensus (K/R)XTQT
motif in each sequence is highlighted with bold
letters.
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Fig. 8.
Mutational analysis of the roles of
individual residues in the (K/R)XTQT motif
in binding to DLC8. A, schematic diagram showing the
point mutations of the GST-fused DIC peptide used in the DLC8 binding
assay. WT, wild type. B, Coomassie Blue
staining of the SDS-PAGE gel showing the interactions between DLC8 and
various GST·DIC peptide mutants. The wild type GST·DIC peptide and
purified GST were used as positive and negative controls, respectively.
The lane numbers in B match the construct numbers in
A. MW, molecular weight markers. C,
binding of a (K/R)XTQT motif-containing peptide (the
9-residue DLC8-binding domain of Bim shown in Fig. 6) to DLC8. In this
figure, the DLC8 dimer is shown in a surface model, and the peptide is
in an explicit atom model. The positively charged amino acids in DLC8
are shown in blue; negatively charged residues are in
red; hydrophobic residues are in yellow; and
polar residues are in gray. The amino acid residues of DLC8
that form intimate electrostatic and hydrogen-bonding interactions are
labeled with amino acid residue numbers and names (the numbers with and
without a prime are used to differentiate residues from two different
monomers of DLC8). The amino acid sequence of the peptide is labeled
with residue name and number using double primes. The coordinates of
the structure were taken from our earlier work (Ref. 18, Protein Data
Bank code 1F95).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit of RNA
polymerase) via residues 138-172 of P protein (21, 22). Sequence
alignments of P proteins from various strains of rabies virus showed
that all contain a conserved KSTQT motif at the center of the protein.
We suggest that DLC8 binds to P protein via this KSTQT motif. In the
protein data base there are a large number of additional viral proteins containing a potential DLC8-binding (K/R)XTQT motif. For
example, poly(A) polymerase catalytic subunit from vaccinia virus
contains a 84KQTQT88 motif. Human
papillomavirus probable E4 protein contains a KQTQT motif in its N
terminus. Several viral proteases (e.g. human adenovirus endoprotease and Haemophilus influenzae
immunoglobulin A1 protease precursor) contain potential DLC8-binding
KSTQT motifs. It is possible that DLC8 is responsible for
dynein-mediated retrograde transport of these viral proteins along
microtubules in infected cells (e.g. retrograde axonal
transport of viral proteins in virus-infected neurons).
-strand structure in the DLC8-target peptide complex (Fig. 8C; see also Ref. 18). To establish a better
structure-function relationship of the (K/R)XTQT
motif-mediated DLC8 binding, the four conserved residues in the motif
were systematically mutated. The structure of the DLC8 complexed with a
(K/R)XTQT motif-containing peptide suggested that the
binding affinity of the peptide to DLC8 is due to a combination of
backbone hydrogen bonding and a number of hydrogen-bonding and
electrostatic interactions between the side chains of the peptide and
DLC8 (18). For example, strong hydrogen-bonding interactions between
the side chain of the Gln residue in the peptide and side chains of
Glu35 and Lys36 of DLC8 were observed. Mutation
of the Gln to Ala, which eliminates the hydrogen-bonding capacity of
the side chain of this amino acid residue, completely abolished peptide
binding to DLC8 (Fig. 8). We believe that the Gln residue plays a
central role in promoting the formation of the peptide-DLC8 complex.
Structure-based sequence alignment of the nNOS peptide and the
(K/R)XTQT motif peptide showed that a Gln residue
in the nNOS peptide occupies the same position as the Gln residue in
the (K/R)XTQT peptide upon formation of complexes with DLC8
(18). The total loss of the interaction observed in the individual Thr
to Gly mutations is probably a combination of the loss of side chain
interactions between the peptide and DLC8 and destabilization of the
backbone
-strand structure of the peptide. Mutations of the Lys
residue to either Ala or Glu lead to somewhat decreased binding
affinity between the peptide and DLC8, suggesting that the Lys residue
in this position has a relatively small contribution to the binding
affinity of the peptide. The mutagenesis study reinforces our earlier
conclusion that the interactions between DLC8 and the
(K/R)XTQT motif-containing peptide are dominated by a
combination of side chain and backbone hydrogen-bonding and
electrostatic interactions (18).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Lap-Chee Tsui for genomic clones of mouse cytoplasmic DIC, Dr. Andreas Strasser for Bim clones used in this study, and Dr. Jim Hackett for careful reading of the manuscript.
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FOOTNOTES |
---|
* This work was partially supported by Grants HKUST6084/98M, 6198/99M, and 6207/00M from the Research Grant Council of Hong Kong to M. Z.) and by the Human Frontier Science Program.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.
¶ Assistant Investigator of the Howard Hughes Medical Institute.
To whom correspondence should be addressed. Tel.:
852-2358-8709; Fax: 852-2358-1552; E-mail: mzhang@ust.hk.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M010320200
2 J.-S. Fan, Q. Zhang, H. Tochio, and M. Zhang, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: DHC, dynein heavy chain; DIC, dynein intermediate chain; DLIC, dynein light intermediate chain; DLC, dynein light chain; PCR, polymerase chain reactions; GST, glutathione S-transferase; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; nNOS, neuronal nitric-oxide synthase; HSQC, heteronuclear single quantum correlation.
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REFERENCES |
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