From the Department of Biochemistry, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China
Received for publication, December 18, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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The minus-ended microtubule motor
cytoplasmic dynein contains a number of low molecular weight light
chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the
dynein complex and its function are largely unknown. Using partially
deuterated, 15N,13C-labeled protein
samples and transverse relaxation-optimized NMR spectroscopic
techniques, the secondary structure and overall topology of Tctex-1
were determined based on the backbone nuclear Overhauser effect pattern
and the chemical shift values of the protein. The data showed that
Tctex-1 adopts a structure remarkably similar to that of the 8-kDa
light chain of the motor complex (DLC8), although the two light chains
share no amino acid sequence homology. We further demonstrated that
Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The
Tctex-1 binding site on DIC was mapped to a 19-residue fragment
immediately following the second alternative splicing site of DIC.
Titration of Tctex-1 with a peptide derived from DIC, which contains a
consensus sequence R/KR/KXXR/K found in various Tctex-1
target proteins, indicated that Tctex-1 binds to its targets in a
manner similar to that of DLC8. The experimental results presented in
this study suggest that Tctex-1 is likely to be a specific cargo
adaptor for the dynein motor complex.
Tctex-1 (t-complex
testis-expressed-1) was originally identified as
a multigene family that maps to the t-complex, a large region of mouse chromosome 17 (known as t-haplotypes)
containing four nonoverlapping inversions that suppress recombination
(1). Male +/t-heterozygotes transmit the mutant chromosome
at >99% frequency to their progeny, a non-Mendelian phenomenon known
as transmission ratio distortion, and male
t/t-homozygotes are completely sterile. The aberrant
expression of Tctex-1 in the t-haplotype mice
(4-fold overexpressed in +/t and 8-fold in t/t)
was suggested to be functionally related to sterility and transmission
ratio distortion (1). This Tctex-1-related meiotic drive hypothesis was
supported by the finding that Tctex-1 is a light chain of cytoplasmic
as well as flagellar inner arm dynein complexes (2, 3).
Cytoplasmic dynein is a microtubule-based molecular motor involved in
various intracelluar motile events including retrograde vesicle
transport, axonal transport, mitotic spindle positioning, and nuclear
migration (4-7). The dynein motor is a multicomponent protein and
contains two heavy chains (~530 kDa), two intermediate chains
(DIC1; ~74 kDa), four light
intermediate chains (~50-60 kDa), and several light chains (DLC; 8, 14, and 22 kDa) (4). Dynein heavy chains directly attach the dynein
complex to microtubules and contain ATPase activity, which is required
for force generation of the motor. DIC is involved in linking the motor
to vesicle-based cargoes by mediating the interaction between dynein
and dynactin (8-10). The functions of other dynein subunits are
largely unknown because of the complexity of the motor complex.
In addition to functioning as a stoichiometric subunit of the
cytoplasmic dynein complex (2, 11), Tctex-1 was also found to interact
with a number of cellular proteins of diverse function. Tctex-1
interacts with the N-terminal region of Doc2, and the interaction
between these two proteins was suggested to be involved in the
dynein-mediated vesicle transport (12). Tctex-1 was also shown to
interact with a 19-residue fragment located at the extreme N terminus
of p59fyn Src family tyrosine protein kinase
(13). Colocalization of Tctex-1 and Fyn at the cleavage furrow and
mitotic spindles in T cell hybridomas undergoing cytokinesis points to
possible roles of dynein in cell cycle control. Interaction of a
lymphocyte surface glycoprotein CD5 with Tctex-1 was suggested to be
linked to internalization of CD5 (14). Additionally, Tctex-1 was
recently reported to interact directly with the cytoplasmic tail of
rhodopsin. This interaction was suggested to be responsible for the
transport of rhodopsin-laden vesicles across the inner segment to the
base of the connecting cilium (15). The discovery of a large number of
functionally unrelated Tctex-1-binding proteins suggests that Tctex-1
is likely to function as an adaptor serving to link specific cargoes to
the dynein motor. Because of limited biochemical and structural
characterization of Tctex-1, the molecular mechanisms governing the
interactions between the protein and its binding partners are unknown.
In this work, we performed a detailed structural characterization of
Tctex-1 by NMR spectroscopic techniques. Using purified recombinant
proteins, we showed that Tctex-1 binds directly to the intermediate
chain of cytoplasmic dynein, and the Tctex-1 binding site was mapped to
a short stretch of amino acid residues in the N-terminal region of DIC.
We further demonstrate that Tctex-1 shares remarkable structural and
target binding similarities with DLC8, although the two light chains
share no amino acid sequence homology.
Construction of Bacterial Expression Plasmids--
The cDNA
encoding mouse Tctex-1 was generously provided by Prof. Yoshimi Takai.
The Tctex-1 expression plasmid was constructed by inserting polymerase
chain reaction-amplified Tctex-1 gene fragment into the NdeI
and BamHI sites of the pET3a vector (Novagen).
The full-length mouse DIC gene was constructed by assembling three
overlapping genomic clones as described in our earlier work (31). The
N-terminal region of DIC (amino acids 1-213) was inserted into the
BamHI and EcoRI sites of the pGEX-4T-1 vector (Amersham Pharmacia Biotech) for expression of a GST fusion protein. Various truncation, deletion, and point mutations of this DIC fragment
were constructed using standard polymerase chain reaction and cloning
techniques. The C-terminal WD repeats (amino acids 214-628) were
constructed as an N-terminal His-tagged protein (31).
Protein Expression and Purification--
Tctex-1 was expressed
by transforming the pET3a vector containing the Tctex-1 gene into
Escherichia coli BL21(DE3) host cells. A single colony of
E. coli cells harboring the expression plasmid was
inoculated into 50 ml of LB with 100 µg/ml ampicillin (LBA). The cell
culture was incubated overnight at 37 °C and then inoculated into 1 liter of fresh LBA medium. Tctex-1 expression was induced by the
addition of isopropyl-1-thio-
Various forms of GST-fused DIC mutant proteins were expressed in
E. coli cells. In a typical purification procedure, cell pellet from 1 liter of culture was resuspended in 25 ml of PBS buffer
(140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, and 1.8 mM
KH2PO4) containing 0.1 mM
phenylmethylsulfonyl fluoride. The GST fusion proteins were purified
following the instructions of the manufacturer (Amersham Pharmacia
Biotech). The purified proteins were dialyzed extensively against PBS
prior to binding experiments.
NMR Sample Preparation--
All NMR samples were prepared by
concentrating purified Tctex-1 using a Centriprep-3 (Amicon)
ultrafiltration device. The protein was exchanged into the desired NMR
sample buffer (50 mM Tris-d11, pH
7.0, 2 mM dithiothreitol-d10, and
10% D2O), with or without 0.4 M KCl, using
ultrafiltration. Preparation of the partially deuterated Tctex-1
followed a protocol described previously with slight modifications
(16). Briefly, a single colony of E. coli cells harboring
the Tctex-1 expression plasmid was inoculated into 50 ml of LBA and
incubated overnight at 37 °C. About 4 ml of this overnight culture
was inoculated into 200 ml of
15N/H2O/[13C]glucose (for
2H,15N,13C triple labeling) or
15N/H2O/[12C]glucose (for
2H,15N double labeling) M9 medium with an
initial A600 value of ~0.04. The cells were
incubated at 37 °C until the A600 value
reached ~0.5. The cells were spun down at 4 °C and then gently
resuspended into 200 ml of 15N, 80% D2O,
[13C]glucose or 15N, 80% D2O,
[12C]glucose M9 medium. The suspension was diluted five
times into 800 ml of 15N, 80% D2O,
[13C]glucose or 15N, 80% D2O,
[12C]glucose M9 medium to give an
A600 value of ~0.1. The cultures were
incubated at 37 °C until the A600 reading
reached ~0.5, and Tctex-1 expression was induced by the addition of
isopropyl-1-thio- NMR Spectroscopy--
All NMR experiments were performed on a
four-channel Varian Inova 750-MHz spectrometer equipped with a
z-axis pulsed field gradient unit and an actively shielded
triple resonance probe. Sequential backbone assignment of Tctex-1 was
completed using three pairs of TROSY-enhanced triple resonance
experiments (17), namely HN(CA)CB and HN(COCA)CB; HNCA and HN(CO)CA;
HNCO and HN(CA)CO (18-20) on an 15N,13C
uniformly, 80% 2H-partially labeled Tctex-1 sample. An
1H-15N HSQC-NOESY experiment (21)
recorded on an 15N-lableled Tctex-1 sample with a mixing
time of 90 ms was used to confirm the sequential assignment. Backbone
HN-HN NOEs were obtained using a 1H-15N
HSQC-NOESY-HSQC experiment (22) recorded on an
15N-uniformly, 80% 2H-partially labeled
Tctex-1 sample with a mixing time of 150 ms. The protein concentrations
of all NMR samples were ~1.0 mM.
GST "Pull-down" Experiments--
For each pull-down assay,
15 µl of a 75% slurry of GST-Sepharose 4B was first washed three
times with 0.5 ml of PBS for equilibration. About 0.1 mg of the GST
fusion proteins (250 µl) was added, and the suspension was agitated
at 4 °C for 1 h. The GST-protein-loaded beads were washed four
times with 0.5 ml of PBS to remove any unbound protein. A total of 100 µl of Tctex-1 (35 µM) solution was added, and the
suspension was agitated at 4 °C for an additional 2 h. The
beads were then washed four times with 0.5 ml of PBS to remove any
unbound Tctex-1 and subsequently boiled with 20 µl of 2 × SDS-polyacrylamide gel sample buffer. The intensities of the Tctex-1
bands on the SDS-polyacrylamide gel were used to judge the
strength of the interaction between Tctex-1 and various GST-DIC fusion proteins.
Competition between DLC8 and Tctex-1 for DIC binding was examined by
adding 100-µl aliquots of DLC8 solutions of increasing concentration
to suspensions of Tctex-1 premixed with GST-DIC-loaded GSH-Sepharose
beads (at a 1:1 molar ratio of Tctex-1 to GST-DIC). The mixtures were
agitated for an additional 2 h, and the beads were subsequently
washed four times with 0.5 ml of PBS. The relative amounts of Tctex-1
and DLC8 pelleted by the GSH-Sepharose beads were used to judge
possible competition between Tctex-1 and DLC8 for DIC binding.
Secondary Structure and Topology of Tctex-1--
To uncover the
molecular basis of Tctex-1's cellular function, we characterized the
structure of the protein in solution by NMR spectroscopic techniques.
An efficient Tctex-1 production and purification procedure was
developed, and large quantities of various forms of stable
isotope-labeled Tctex-1 were obtained for NMR structural studies. The
1H-15N HSQC spectrum of 15N-labeled
Tctex-1 showed that the majority of the backbone resonances of the
protein were extraordinary broad at various protein concentrations tested (0.1-1 mM). The severe line broadening is likely to
be a combined effect of slow to intermediate time scale conformational exchange and nonspecific protein aggregation (for more detail, see
below). An array of triple resonance experiments on
15N,13C-labeled Tctex-1, aiming to obtain the
backbone assignment of the protein, failed. Both 15N- and
13C-separated three-dimensional NOESY experiments of
Tctex-1 showed unusually low amounts of NOE cross-peaks. To circumvent
sample aggregation and conformational exchange-induced T2 shortening, partial deuteration (23, 24) of the protein (~80%) together with
TROSY techniques (17) were used for NMR characterization of Tctex-1 in
solution. Using a combination of TROSY-based HNCA, HN(CO)CA, HN(CA)CB,
and HN(COCA)CB triple resonance experiments on a
2H,13C,15N-triple labeled Tctex-1
sample, we were able to obtain essentially complete backbone resonance
assignment of the protein. A 15N-separated
three-dimensional NOESY experiment recorded on a
15N-labeled Tctex-1, and an HSQC-NOESY-HSQC experiment
recorded on a 2H,15N-labeled protein sample
were used to confirm the assignment obtained by the triple resonance
experiments. Fig. 1A shows the
TROSY-HSQC spectrum of Tctex-1 with each backbone amide resonance
labeled with amino acid residue name and number. The completion of the backbone chemical shift assignment allowed us to determine the secondary structure of Tctex-1 using a slightly modified chemical shift
index approach (25). As both 13C
Next, we determined the overall topology of Tctex-1. To obtain maximal
backbone amide resolution, we used an HSQC-NOESY-HSQC experiment
recorded on 2H,15N-labeled Tctex-1 to detect
backbone HN-HN NOEs. Large numbers of long range, inter- Tctex-1 Binds to a Small Peptide Fragment at the N-terminal
End of DIC--
Earlier biochemical experiments showed that
Tctex-1 and DIC cofractionated from a KI-treated cytoplasmic dynein
complex preparation, suggesting that Tctex-1 and DIC might interact
directly with each other (2, 11). We used purified recombinant proteins
to test direct interaction between Tctex-1 and DIC. A GST-fused
fragment spanning residues 1-213 of DIC was found to interact robustly with Tctex-1 in the GSH-Sepharose pull-down assay (Fig.
3B, lane 3),
indicating that Tctex-1 and DIC can indeed interact with each other
directly. As expected, purified, recombinant full-length DIC was also
able bind to Tctex-1 (data not shown). In contrast, the C-terminal part
of DIC which contains the highly conserved Trp-Asp repeats failed to
bind to Tctex-1 (data not shown). We then mapped the exact Tctex-1
binding site of DIC by creating a series of DIC truncation mutations
(Fig. 3A). A 19-residue fragment (residues 124-142) of DIC
was identified as sufficient for binding to Tctex-1 (for the amino acid
sequence of the peptide fragment, see Fig. 3A).
Given the overall structural similarities between Tctex-1 and DLC8, we
suspected that Tctex-1 might also bind to a short peptide fragment, as
does DLC8 (27). To localize the precise Tctex-1 binding site further,
we deleted a positive charged, 5-residue fragment (RRLHK) located at
the N-terminal end of the 19-residue Tctex-1 binding fragment of DIC
(Fig. 4). Deletion of the 5-residue fragment completely abolished the interaction between DIC and Tctex-1,
indicating that this 5-residue cassette plays an important role in
supporting the Tctex-1·DIC complex formation. However, this
5-residue fragment alone is not sufficient for effective binding to
Tctex-1 because a GST fusion peptide containing this 5-residue fragment
plus a few amino acid residues at both ends was not able to pull down
Tctex-1 (Fig. 4B, lane 5). These data indicate
that both the N- and C-terminal parts of the 19-residue DIC fragment
are required for the Tctex-1·DIC complex formation.
DIC Binds to the Mutational Analysis of the Consensus Tctex-1 Binding
Sequence--
To assess the potential roles of the conserved amino
acids in the consensus R/KR/KXXR/K sequence of DIC in
Tctex-1 binding, we performed a systematic mutational analysis. The
data indicate that no single residue in the consensus sequence (RRLHK)
plays a dominant role in supporting the Tctex-1·DIC complex formation (Fig. 7). The second arginine residue in
the consensus sequence appears to make a slightly larger contribution
to the complex formation (Fig. 7B, lane 5).
However, deletion of the whole consensus sequence resulted in complete
disruption of the Tctex-1·DIC complex (Fig. 4), suggesting that these
amino acid residues may function additively in binding to Tctex-1.
The DIC truncation experiment showed that residues C-terminal
to the consensus RRLHK sequence (i.e. LGVSKVTQVDFL) are also involved in Tctext-1 binding (Fig. 4). Consistent with this, mutation of Leu-131 to Asn greatly diminished the interaction between Tctex-1 and DIC (Fig. 8, lane 9).
However, a combination of the consensus RRLHK sequence and Leu-131 is
not sufficient for strong binding because the GST-fused peptide
containing both the consensus sequence and Leu-131 was not able to form
a stable complex with Tctex-1 (Fig. 5, lane 5). Further
mutational analysis will be required to identify the structural
features that are essential for Tctex-1 binding.
The Tctex-1 and DLC8 Binding Sites in DIC Are Mutually
Independent--
The Tctex-1 binding site (residues 124-142,
LGRRLHKLGVSKVTQVDFL) identified in this study and the DLC8 binding site
(151-155, KETQT) in DIC are immediately next to each other in the
amino acid sequence of DIC (Fig. 9, and
Ref. 31). Additionally, the Tctex-1 binding site contains a KVTQV
sequence that is similar to the KETQT DLC8 binding motif (31).
Therefore, there is a possibility that the binding of Tctex-1 and DLC8
on DIC are mutually exclusive. To test this possibility, we performed a
binding competition experiment as shown in Fig. 8. In this experiment,
equal molar amounts of GST-DIC and Tctex-1 were mixed with increasing
molar ratio amounts of DLC8 (Fig. 8). The GST-DIC·Tctex-1·DLC8
ternary complex was pelleted by GSH-Sepharose beads and subsequently
analyzed by SDS-polyacrylamide gel electrophoresis. Data in Fig. 8
clearly demonstrate that excess DLC8 does not displace Tctex-1 from
DIC, indicating that Tctex-1 and DLC8 can bind simultaneously to DIC. In a reverse experiment, we found that excess Tctex-1 does not compete
with DLC8 for binding to DIC, consistent with our conclusion that the
Tctex-1 binding site and the DLC8 binding site on DIC are mutually
independent (data not shown).
As a part of our continuing effort to understand the structure and
function of cytoplasmic DLCs, we performed a detailed structural analysis of Tctex-1 in this study. Using multidimensional
TROSY-enhanced NMR spectroscopic techniques, we determined the
secondary structure and the topology of Tctex-1 in solution. Tctex-1
shares a remarkably similar structure with DLC8 both at the secondary
structure and folding topology levels (26, 27), although the two
proteins display very limited amino acid sequence homology.
Analogous to DLC8, both gel filtration and chemical cross-linking
studies showed that Tctex-1 can exist as a dimer in solution (data
not shown). A full structural determination of Tctex-1 and/or its
complex with a target peptide is required to answer how the Tctex-1
dimer is assembled in solution. Detailed comparisons of the
structures of Tctex-1 and DLC8, both in apo- and target-bound
forms, will help to elucidate how the two structurally similar light
chains distinguish their respective targets.
In DLC8, target peptides bind to the protein by augmenting the
Although the amino acid residues downstream of the consensus
R/KR/KXXR/K sequence are necessary for effective binding of
Tctex-1 and its targets, sequence alignment analysis shows that no
obvious homology can be observed in these regions (Fig. 6). The
sequence downstream of the consensus R/KR/KXXR/K sequence in
DIC is a mix of hydrophobic and hydrophilic residues (LGVSKVTQV). The
same region in CD5 is composed mainly of hydrophilic residues; only one
hydrophobic residue occurs (KFRQKKQRQ). It seems that Tctex-1 is
capable of binding to a number of targets with diverse amino acid
sequences, a phenomenon that was also observed for DLC8 (27). Mutational analysis indicated that polar/charged interactions between
the positively charged consensus sequence R/KR/KXXR/K and
negatively charged amino acids from Tctex-1 are likely to play
important roles in binding specificity because deletion of all three
positively charged residues of DIC abolished its binding to Tctex-1.
Hydrophobic interactions between amino acid residues downstream of the
consensus sequence and Tctex-1 are also expected to provide favorable
binding energy for complex formation, as mutation of Leu-131 of DIC to
Asn greatly reduced its affinity for Tctex-1 (Fig. 7).
Because of alternative splicing of the two DIC genes, there at least
five different isoforms of DIC in mammals (8, 10, 28, 29). Fig. 9 shows
a schematic diagram of the domain organization of DIC. The function of
the C-terminal Trp-Asp repeats is still unknown. The high amino acid
sequence homology of the Trp-Asp repeats domain of DICs from both
cytoplasmic and axonemal DICs suggests that this domain is likely to be
responsible for attaching DICs to the heavy chains of the motor
complex. The N-terminal ~120 residues (including the two alternative
splicing sites and the Ser-rich region) of DIC was identified to
interact with dynactin. The discovery of the interaction between DIC
and dynactin suggested that DIC functions as the adaptor to link the
motor complex to its vesicle-based cargoes (8). Following the
dynactin binding region of DIC are the Tctex-1 and DLC8 binding
regions. Because both light chains are capable of binding a large
number of functional unrelated proteins, it is likely that the light
chains function as motor adaptors for transporting various specific
cargoes. This hypothesis is supported by the observation that the two
target binding sites of DLC8 (and also likely Tctex-1) can
simultaneously bind to DIC and one of its target
proteins.2 Unlike kinesins
and myosins that contain a large number of isoforms, cytoplasmic dynein
contains two copies of identical heavy chains with motor activities. It
is likely that a combination of various light chains and light
intermediate chains as well as intermediate chains allows a single
cytoplasmic dynein complex to move a vast number of molecular cargoes
along microtubules.
The dynactin binding region of DIC contains alternative splicing sites
and potential protein phosphorylation sites within the Ser-rich region
(Fig. 9). The dynein light chain binding domains in DIC are situated
immediately C-terminal to the second alternative splicing region. This
means that neither alternative splicing nor phosphorylation of DIC
would have any effect on Tctex-1 and DLC8 binding on DIC. A competition
binding assay of Tctex-1 and DLC8 on DIC showed that the bindings of
Tctex-1 and DLC8 on DIC are mutually independent (Fig. 8), suggesting
that both Tctex-1 and DLC8, together with their unique cargoes, can
bind to DIC simultaneously. Cytoplasmic dynein contains another light
chain called RP3, which shares 52% amino acid identity to Tctex-1.
However, RP3 displays distinct target binding properties when compared with Tctex-1. In contrast to what was observed for Tctex-1, RP3 does
not interact with rhodopsin (15) or Doc2 (12). Tctex-1 and RP3 are
regulated differentially in both a developmental and tissue-specific
manner. Functionally distinct populations of cytoplasmic dynein may
contain different Tctex-1 family light chains. Subcellular localization
of specific light chains, including Tctex-1, may also contribute to the
functional differences of dynein complexes (30). We do not know whether
RP3 directly binds to DIC, or if it does, whether Tctex-1 and RP3 bind
to DIC in a mutually exclusive manner. Further work is required to
address these questions. We further note that although the amino acid
sequence of the DLC8 and Tctex-1 binding region shown in Fig. 9 is
highly conserved in cytoplasmic DIC, these sequences are not clearly
identifiable in axonemal DICs, suggesting a possible difference in the
assembly of the light chains between axonemal and cytoplasmic dyneins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside when the A600 of the culture reached ~0.6.
Pelleted cells from 2 liters of culture were resuspended in 50 ml of
buffer A (50 mM Tris-HCl, pH 7.9, 5 mM
-mercaptoethanol, and 1 mM EDTA) containing 0.1 mM phenylmethylsulfonyl fluoride and lysed by sonication.
The lysate was centrifuged at 18,000 rpm (Sorvall SS34 rotor) for 30 min at 4 °C, and the supernatant was loaded onto a 50-ml
DEAE-Sepharose Fast Flow column (Amersham Pharmacia Biotech). The
column was washed with buffer A until the A280
reading was steady, and the column was then eluted with 200 ml of
buffer A with a linear NaCl gradient of 0-0.3 M. Fractions
containing Tctex-1 were pooled and concentrated to about 8 ml before
loading onto a Sephacryl-100 (Amersham Pharmacia Biotech) gel
filtration column preequilibrated with buffer A containing 0.5 M NaCl. The eluted fractions containing Tctex-1 were pooled
and dialyzed against buffer A before loading onto a Mono Q HR 10/10
column (Amersham Pharmacia Biotech). The Mono Q column was washed with
buffer A, and Tctex-1 was eluted using 70 ml of the same buffer with an
NaCl gradient of 0-0.35 M. The purified Tctex-1 was
dialyzed against buffer A and stored at
80 °C.
-D-galactopyranoside to a final
concentration of 0.5 mM. The bacterial culture was incubated overnight at 16 °C before the cells were harvested.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and 13C
shifts are sensitive to secondary structure, and as they shift to
opposite fields in a given secondary structure, a combined 13C
/13C
secondary shift presented by
subtracting the 13C
secondary shift of a residue from
its 13C
secondary shift enhances secondary
structure-induced chemical shift changes of the protein (Fig.
1B). Presentation of the secondary structure chemical shifts
using combined 13C
/13C
shifts has the
additional advantage of canceling potential secondary shift errors
resulted from chemical shift referencing. Based on the data in Fig.
1B, we conclude that Tctex-1 is composed of two long
N-terminal
-helices (
1 and
2, with starting and ending
residues labeled in Fig. 1B) followed by four
-strands (
1 to
4). The secondary structure of Tctex-1 derived from the chemical shift data was supported by the backbone NOE patterns of the
protein derived from the two three-dimensional 15N-NOESY
experiments (HSQC-NOESY-HSQC and 15N-separate NOESY, data
not shown). The chemical shift values of the residues (Asp-3 to Thr-10)
N-terminal to
1 indicate that this fragment of Tctex-1 appears to
assume a random coil-like structure. The random coil structure of the
N-terminal fragment inferred from chemical shift data is also supported
by the exceptionally narrow line widths of these residues and the lack
of detectable long range backbone NOE of the region. The secondary
structure of Tctex-1 shown in Fig. 1B is remarkably similar
to that of another dynein light chain, DLC8 (26, 27), although the two
light chains have very limited amino acid sequence homology.
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Fig. 1.
Sequential assignment and the secondary
structure of Tctex-1. Panel A,
1H,15N-TORSY-HSQC spectrum of 15N-,
80% 2H-labeled Tctex-1 in 50 mM
Tris-d11, pH 7.0, 2 mM
dithiothreitol-d10, 0.4 M KCl at
22 °C. The assignment of each backbone amide resonance is labeled
with the amino acid residue name and number. The crowded region of the
spectrum (the boxed region at the center) is
magnified and shown as an inset at the upper left
corner of the spectrum. Panel B, combined
13C /13C
chemical shift index plot of
Tctex-1. In this plot, the secondary chemical shift of each amino acid
residue is expressed as the 13C
secondary shift minus
the 13C
secondary shift with a smoothing factor of 3. The secondary structure of Tctex-1 is shown at the top of
the graph. Amino acid residues 4-7 may adopt a
-strand-like
structure based on relatively small deviation of the combined secondary
chemical shifts in this region.
-strand NOEs
were used to determine the folding topology of the Tctex-1
-strands
(Fig. 2). The four
-strands of Tctex-1
form an antiparallel
-sheet structure with a
3-
4-
1-
2 arrangement (Fig. 2). Several unambiguous inter-
-helical HN-HN NOEs
spanning the entire helices (e.g. backbone amide NOEs
between Asp-15 and Thr-55, Ile-20 and Thr-50, and Gly-30 and Val-39)
indicate that the two
-helices of Tctex-1 are antiparallel to each
other. The detection of long range backbone amide NOEs between amino acid residues from the
-helices and
-strands (e.g.
NOEs between Ala-28 and Cys-104, Asn-32 and Trp-97, Asn-45 and Cys-67,
and Leu-54 and Asp-86) indicate that the two helices pack against the
antiparallel
-sheet of the protein (see Fig. 6, inset).
The overall topology of Tctex-1 is again very similar to that of DLC8 (26, 27).
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Fig. 2.
The topology of the
-sheet of Tctex-1. The long range,
inter-
-strand HN-HN NOEs (shown with black arrows) were
used to determine the topology of the four
-strands of Tctex-1. The
dotted lines are used to indicate expected inter-
-strand
hydrogen bonds. The side chains of amino acid residues highlighted with
shaded circles are on the same side (relative to the
-sheet plane) as the two
-helices of the protein.
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Fig. 3.
Mapping of the Tctex-1 binding domain of
DIC. Panel A, schematic diagram showing the
various truncation mutants of GST-fused DIC used to map the Tctex-1
binding domain. The binding of each mutant with Tctex-1 is also
summarized in the figure. Panel B, Coomassie Blue
staining of an SDS-polyacrylamide gel showing the interaction between
various purified DIC fragments and Tctex-1. The lane number
in panel B corresponds to the construct number in
panel A. Purified Tctex-1 (lane 1) was used as a
protein marker, and pure GST (lane 2) was a negative
control.
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Fig. 4.
Dissection of the Tctex-1 binding domain of
DIC. A, schematic diagram showing the truncation and
deletion mutants of GST-DIC used to analyze the interaction between
Tctex-1 and DIC. The amino acid sequence of constructs 5 and
6 are shown in the figure. The consensus RRLHR sequence is
highlighted with boldface letters. Panel B,
Coomassie Blue staining of an SDS-polyacrylamide gel showing the
interaction between Tctex-1 and various GST-DIC fusion proteins. The
lane number in panel B corresponds to the
construct number in panel A. Purified Tctex-1
(lane 1) was used as a protein marker, and pure GST
(lane 2) was a negative control.
2-Strand and the
2/
3-Loop of
Tctex-1--
We next set out to identify the DIC binding region
in Tctex-1. To simplify the experiment, we used a synthetic peptide
corresponding to the full-length Tctex-1 binding domain of DIC to
titrate with partially deuterated 15N-labeled Tctex-1.
Unfortunately, the 1H-15N HSQC of Tctex-1 in
the presence of this peptide became exceedingly broad and beyond
interpretation, suggesting a severe aggregation of the protein-peptide
complex (data not shown). Inspection of the amino acid sequences of a
number of Tctex-1 binding regions in various targets revealed a short
stretch of consensus sequence of R/KR/KXXR/K (Fig.
5). Although an 11-residue DIC peptide
containing the consensus sequence fused to GST was not able to pull
down Tctex-1 (the assay requires high affinity binding between the two
proteins; Fig. 3), we anticipated that NMR titration of Tctex-1 with
the same 11-residue peptide would allow us to detect the possible
interaction between the protein and peptide (even if the interaction
were relatively weak). To test this possibility, we titrated
15N-labeled Tctex-1 with the 11-residue synthetic peptide
(LGRRLHKLGVS from residue 124 to 134 of DIC, Fig. 4). Significant
chemical shift changes of a number of backbone amide cross-peaks of
Tctex-1 were observed during the titration, indicating that the
11-residue DIC peptide can indeed bind to the protein. Fig.
6 shows an overlay plot of the HSQC
spectra of Tctex-1 at the start (blue) and end (red) points of the titration. The amino acid residues that
display significant chemical shift changes are labeled, and these amino acid residues are likely to be those involved in the peptide binding. The inset of Fig. 6 shows the distribution of the amino
acids that undergo significant peptide-induced chemical shift changes, and these residues are found mainly at the end of
2 (Cys-83, Phe-84,
Trp-85, and Asp-86) and the
2/
3-loop (Thr-89, Asp-90, and Gly-91)
of Tetex-1. In addition, several residues that are in close proximity
to this region (e.g. Thr-55 and Lys-56 at the end of
1,
and Phe-61 and Lys-62 at the start of
1) also experience some
chemical shift changes. Based on the data in Fig. 6, we conclude that
the
2-strand and the
2/
3-loop are the main DIC binding region
in Tctex-1.
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Fig. 5.
Sequence alignment analysis of selected
Tctex-1 binding domains. Amino acid sequence alignment of the
Tctex-1 binding domains of DIC (this work), DOC2 and DOC2
(12),
CD5 antigen (14), and peropsin reveals a consensus
R/KR/KXXR/K motif in Tctex-1 binding domains (shown in
boldface letters). However, no obvious consensus sequence
can be observed in the Tctex-1 binding domain of
p59fyn (13) and rhodopsin (15).
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Fig. 6.
Interaction of Tctex-1 with
a DIC peptide. The figure shows an overlay plot of TROSY-HSQC
spectra of Tctex-1 at the starting (blue) and end
(red) points of titration with an 11-residue peptide
(LGRRLHKLGVS) corresponding to the N-terminal part of the Tctex-1
binding domain of DIC. The concentration of Tctex-1 (15N-,
80% 2H-labeled) is 0.4 mM, and the protein was
dissolved in 50 mM Tris-d11, pH 7.0, 2 mM dithiothreitol-d10. The molar
ratio of the DIC peptide to Tctex-1 at the final point of the titration
is 2:1. The residues that show significant chemical shift changes upon
the addition of the DIC peptide are marked with open boxes,
and these amino acid residues are mapped to the topology structure of
Tctex-1 shown as an inset in the figure. The dashed
lines in the topology diagram of Tctex-1 are used to indicate the
uncertainty of the connections of the -strands due to potential
domain swapping of the Tctex-1 dimer.
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Fig. 7.
Mutational analysis of the consensus Tctex-1
binding motif. Panel A, schematic diagram showing the
individual mutations of the DIC fragment created in the experiment. The
binding of each mutant with Tctex-1 is also summarized in the figure.
WT, wild type. Panel B, Coomassie Blue staining
of an SDS-polyacrylamide gel showing the interaction between various
DIC mutants and Tctex-1. The lane number in panel
B corresponds to the construct number in panel
A. Purified Tctex-1 (lane 1) was used as a protein
marker, and pure GST (lane 2) was a negative control.
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Fig. 8.
Tctex-1 and DLC8 can simultaneously bind to
DIC. Coomassie Blue staining of an SDS-polyacrylamide gel showing
that excess DLC8 does not compete with Tctex-1 for binding to DIC.
Lanes 3-9 show Tctex-1 and DLC8 pulled down by GST-DIC. The
molar ratios of DLC8 (relative to Tctex-1 and GST-DIC, which were
premixed at an equal molar ratio) used in the
dose-dependent competition experiment are indicated at the
top of each lane. Lanes 1 and
2 are purified Tctex-1 and DLC8 protein markers.
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Fig. 9.
Summary of the domain organization of
cytoplasmic DIC. Cytoplasmic DIC contains an N-terminal
coiled-coil domain that binds to dynactin complex followed by
alternative splicing sites and a highly conserved Ser-rich region
sandwiched by the two alternative splicing sites. Immediately following
the second alternative splicing site are the nonoverlapping Tctex-1 and
DLC8 binding sites. The C-terminal Trp-Asp repeat is expected to form
-propeller structures, and this domain is likely responsible for
binding to the heavy chain of the dynein complex. A partial amino acid
sequence alignment of various isoforms of cytoplasmic DIC including the
alternative splicing sites, Ser-rich region, and the two light chain
binding regions is also included in the figure. The Tctex-1 and DLC8
binding sites of DIC are highlighted with boldface letters,
and these two regions are highly conserved.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheet via the
2-strand in an antiparallel fashion (26, 27). Our
NMR studies indicated that Tctex-1 binds a target peptide derived from
DIC using a mechanism similar to that of DLC8 (i.e. the
2-strand and the
2/
3-loop are the major target binding regions
for both proteins) (Fig. 6). It is possible that the short DIC peptide
(LGRRLHKLGVS) used in the NMR titration experiment may also bind to
Tctex-1 by pairing with the
2-strand of the protein. However, we
also notice significant differences between Tctex-1 and DLC8 in their
respective target bindings. For example, DLC8 is capable of binding to
a KXTQT motif-containing peptide, which is as short as 9 residues, with
high affinity and specificity (27, 31). In contrast, the
Tctex-1-binding peptide is likely to be significantly longer. An
11-residue DIC peptide containing a consensus R/KR/KXXR/K
sequence with a few amino acid residue extensions at both ends was not
able to bind to Tctex-1 with high affinity (Fig. 5). To achieve high
affinity binding, an additional ~10 amino acids C-terminal to the
RRLHK sequence of DIC are required. Given the length of the
Tctex-1-binding peptide, the target binding site on Tctex-1 is likely
to involve regions other than the
2-strand and the
2/
3-loop of
the protein. Unfortunately, because of the poor behavior of Tctex-1
complexed with a synthetic peptide containing the complete Tctex-1
binding region of DIC in solution, we were not able to determine the
exact regions of Tctex-1 which are involved in target binding.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. Yoshimi Takai for providing the Tctex-1 gene and Dr. Jim Hackett for careful reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants HKUST6084/98M, 6198/99M, and 6207/00M from the Research Grants Council of Hong Kong and the Human Frontier Science Program (to M. Z.). The NMR spectrometer used in this work was purchased using funds donated to the Biotechnology Research Institute of HKUST by the Hong Kong Jockey Club.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.
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.M011358200
2 K. W.-H. Lo, and M. Zhang, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: DIC(s), dynein intermediate chain(s); DLC, dynein light chain; GST, glutathione S-transferase; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum correlation; TROSY, transverse relaxation-optimized NMR spectroscopy.
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REFERENCES |
---|
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---|
1. | Lader, E., Ha, H. S., O'Neill, M., Artzt, K., and Bennett, D. (1989) Cell 58, 969-979[CrossRef][Medline] [Order article via Infotrieve] |
2. |
King, S. M.,
Dillman, I. I. I., J. F.,
Benashski, S. E.,
Lye, R. J.,
Patel-King, R. S.,
and Pfister, K. K.
(1996)
J. Biol. Chem.
271,
32281-32287 |
3. |
Harrison, A.,
Olds-Clarke, P.,
and King, S. M.
(1998)
J. Cell Biol.
140,
1137-1147 |
4. | King, S. M. (2000) Biochim. Biophys. Acta 1496, 60-75[Medline] [Order article via Infotrieve] |
5. | Karki, S., and Holzbaur, E. L. (1999) Curr. Opin. Cell Biol. 11, 45-53[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Hirokawa, N.
(1998)
Science
279,
519-526 |
7. | Vallee, R. B., and Sheetz, M. P. (1996) Science 271, 1539-1544[Abstract] |
8. | Vaughan, K. T., and Vallee, R. B. (1995) J. Cell Biol. 131, 1507-1516[Abstract] |
9. |
Ma, S.,
Trivinos-Lagos, L.,
Graf, R.,
and Chisholm, R. L.
(1999)
J. Cell Biol.
147,
1261-1273 |
10. | Susalka, S. J., Hancock, W. O., and Pfister, K. K. (2000) Biochim. Biophys. Acta 1496, 76-88[Medline] [Order article via Infotrieve] |
11. | King, S. M., Barbarese, E., Dillman, I. I. I., J. F., Benashski, S. E., Do, K. T., Patel-King, R. S., and Pfister, K. K. (1998) Biochemistry 37, 15033-15041[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Nagano, F.,
Orita, S.,
Sasaki, T.,
Naito, A.,
Sakaguchi, G.,
Maeda, M.,
Watanabe, T.,
Kominami, E.,
Uchiyama, Y.,
and Takai, Y.
(1998)
J. Biol. Chem.
273,
30065-30068 |
13. |
Campbell, K. S.,
Cooper, S.,
Dessing, M.,
Yates, S.,
and Buder, A.
(1998)
J. Immunol.
161,
1728-1737 |
14. | Bauch, A., Campbell, K. S., and Reth, M. (1998) Eur. J. Immunol. 28, 2167-2177[CrossRef][Medline] [Order article via Infotrieve] |
15. | Tai, A. W., Chuang, J.-Z., Bode, C., Wolfrum, U., and Sung, C.-H. (1999) Cell 97, 877-887[Medline] [Order article via Infotrieve] |
16. | Rosen, M. K., Gardner, K. H., Willis, R. C., Parris, W. E., Pawson, T., and Kay, L. E. (1996) J. Mol. Biol. 263, 627-636[CrossRef][Medline] [Order article via Infotrieve] |
17. | Pervushin, K., Braun, D., Fernandez, C., and Wuthrich, K. (2000) J. Biomol. NMR 17, 195-202[CrossRef][Medline] [Order article via Infotrieve] |
18. | Kay, L. E., and Gardner, K. H. (1997) Curr. Opin. Struct. Biol. 7, 722-731[CrossRef][Medline] [Order article via Infotrieve] |
19. | Kay, L. E. (1997) Biochem. Cell Biol. 75, 1-15[CrossRef][Medline] [Order article via Infotrieve] |
20. | Clore, G. M., and Gronenborn, A. M. (1998) Trends Biotechnol. 16, 22-34[CrossRef][Medline] [Order article via Infotrieve] |
21. | Zhang, O., Forman-Kay, J. D., Shortle, D., and Kay, L. E. (1997) J. Biomol. NMR 9, 181-200[CrossRef][Medline] [Order article via Infotrieve] |
22. | Ikura, M., Bax, A., Clore, G. M., and Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 9020-9022 |
23. | Grzesiek, S., Anglister, J., Ren, H., and Bax, A. (1993) J. Am. Chem. Soc. 115, 4369-4370 |
24. | Yamazaki, T., Lee, W., Arrowsmith, C. H., Muhandiram, D. R., and Kay, L. E. (1994) J. Am. Chem. Soc. 116, 11655-11666 |
25. | Wishart, D. S., Sykes, B. D., and Richards, F. M. (1992) Biochemistry 31, 1647-1651[Medline] [Order article via Infotrieve] |
26. | Liang, J., Jaffrey, S. R., Guo, W., Snyder, S. H., and Clardy, J. (1999) Nat. Struct. Biol. 6, 735-740[CrossRef][Medline] [Order article via Infotrieve] |
27. | Fan, J.-S., Zhang, Q., Tochio, H., Li, M., and Zhang, M. (2001) J. Mol. Biol. 306, 97-108[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Nurminsky, D. I.,
Nurminskaya, M. V.,
Benevolenskaya, E. V.,
Shevelyov, Y. Y.,
Hartl, D. L.,
and Gvozdev, V. A.
(1998)
Mol. Cell. Biol.
18,
6816-6825 |
29. | Crackower, M. A., Sinasac, D. S., Xia, J., Motoyama, J., Prochazka, M., Rommens, J. M., Scherer, S. W., and Tsui, L. C. (1999) Genomics 55, 257-267[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Tai, A. W.,
Chuang, J.-Z.,
and Sung, C.-H.
(1998)
J. Biol. Chem.
273,
19639-19649 |
31. |
Lo, K. W.-H.,
Naisbitt, S.,
Fan, J.-S.,
Sheng, M.,
and Zhang, M.
(2001)
J. Biol. Chem
276,
14059-14066 |