From the European Molecular Biology Laboratory
(EMBL), Hamburg Outstation, EMBL c/o Deutsches Electronen-
Synchrotron (DESY), Notkestrasse 85, D-22603 Hamburg, Germany, the
§ Randall Centre, New Hunt's House, King's College London,
Guy's Campus, London, SE1 1UL, United Kingdom, the
EMBL,
Meyerhofstrasse 1, D-69117 Heidelberg, Germany, and the
§§ Institute of Crystallography, Russian Academy
of Sciences, Leninsky pr. 59, 117333 Moscow, Russia
Received for publication, October 5, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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Telethonin interacts specifically with the
two Z-disk IG-like domains (Z1Z2) at the N terminus of titin,
the largest presently known protein. Analytical ultracentrifugation and
synchrotron radiation x-ray scattering were employed to study the
solution structures of Z1Z2 and its complexes with telethonin, and low resolution models were constructed ab initio from the
scattering data. A seven residues-long polyhistidine tag was localized
at the tip of the Z1 domain by comparison of independent models of native and His-tagged versions of Z1Z2. The stoichiometry and shape of
the complex between the telethonin construct lacking the C terminus and
Z1Z2 indicate antiparallel association of two Z1Z2 molecules with
telethonin acting as a central linker. The complex of full-length
telethonin with Z1Z2 appears to also have a 1:2 stoichiometry at
concentrations below 1 mg/ml but dimerizes at higher
concentrations. These results suggest a possible role of
telethonin in linking titin filaments at the Z-disk periphery.
The giant muscle protein titin is the largest gene product found
in the human genome, with a molecular mass
(MM)1 in the range of 3-4
MDa depending on differential spliced variants (1, 2). In one of
its longest isoforms, vertebrate-striated muscle titin contains more
than 38,000 residues organized in 363 exons (2). Titin spans across
one-half of a sarcomere in striated and cardiac muscle cells. Its N
terminus is located within the Z-disc defining the border between
adjacent sarcomeres, whereas its C terminus is located in the M-line at
the center of the sarcomere. Titin acts as a molecular ruler within the
sarcomeric units by providing many spatially defined binding sites for
other sarcomeric proteins over the distance of an entire
half-sarcomere (3).
The Z-disk has a thickness between 40 and 100 nm depending on muscle
type as revealed by electron microscopy (4). It functions as a terminal
anchor for a number of sarcomere filament systems like titin, actin,
and nebulin. These filaments are cross-linked by a complex network of
protein components, including The N termini of titin cross the center of the Z-disk, allowing a range
of about 500 residues of titin to be integrated into the Z-disk (11).
Immunofluorescence analysis has revealed a symmetric distribution of
the N terminus of titin at the periphery of the Z-disk (10, 11).
Co-localization studies and yeast two-hybrid analysis revealed tight
binding of the N terminus of titin via its two N-terminal
immunoglobulin-like domains Z1 and Z2 to telethonin, also referred to
as T-cap (11, 12). Telethonin is a small protein composed of 167 residues with unknown fold (13). It bears a specific phosphorylation
site at its C terminus, which is a substrate for the muscle serine
kinase of titin (14). The physiological function of telethonin is,
however, still unclear. Besides a possible role in myofibril assembly,
a more dynamic role in myofibril turnover is emerging (15, 16).
Telethonin thus seems to play a role in developmental and functional regulation.
Truncation analysis revealed that the N terminus of telethonin is
sufficient for binding to the N terminus of titin (11). Having
identified telethonin as a specific ligand of the N terminus of titin
it becomes a key marker for positioning the N terminus of titin in the
Z-disk. There is also increasing evidence that mutations both in the
binding segments of the N terminus of titin and telethonin lead to
severe pathological disorders like limb-girdle muscle dystrophy and
cardiomyopathy (17, 18). The late onset of both pathologies is also in
agreement with a role in later stages of muscle development. To provide
molecular insight into this protein-protein interaction,
Z1Z2-telethonin complexes with two different telethonin constructs were
prepared. The first construct is the truncated telethonin segment
minimally required for binding to titin (11), whereas the second one
has the full-length telethonin sequence. The structure and
stoichiometry of Z1Z2 and of the two complexes in the solution were
studied by small angle scattering. This method reveals the low
resolution structure of biological macromolecules in nearly native
conditions (19) and was, in particular, successfully employed to study
muscle proteins in solution (20, 21). In the present study, synchrotron
small-angle x-ray scattering (SAXS) data were collected and analyzed
using recently developed ab initio methods (22, 23). The
results point to a cross-linking function of telethonin within the
Z-disk of muscle sarcomeres.
Strains and Plasmids--
All plasmids were constructed in
Escherichia coli Strain DH5 Yeast Two-hybrid Analysis and Constructs--
IG domains from
the titin N terminus were cloned into a modified pLexA vector described
previously (10), and interactions with full-length telethonin in pGAD10
(clone 633 from a screen with Z1Z2, Ref. 12) were monitored in L40
cells (25) as described (10). Domain nomenclature followed the human
cardiac titin sequence (EMBL X90568, Ref. 1). Construct boundaries were
as follows: Z1, residues 1-102; Z1Z2, residues 1-194; Z2, residues
104-194; Z1-X, residues 1-112; and X-Z2, residues 90-194. Telethonin
truncations were designed based on structure analysis, generated by
PCR, and subcloned into pGAD10. Truncation constructs of telethonin
comprised the following residues: deltaC1 105-167, deltaC2 86-167,
deltaN1 1-105, deltaN2 1-137, and delta N3 1-90. Their interaction
with titin was analyzed by co-transformation with pLexA-Z1Z2 into L40 cells as described (12).
Mutagenesis--
The QuikChangeTM site-directed
mutagenesis protocol (Stratagene) was used to introduce the point
mutations into telethonin (C18S, C47S, and C127S) using mutagenic
oligonucleotides of 30-35 bases in length (MWG Biotech, Ebersberg,
Germany). Cys-8 and Cys-15, which are close to the N
terminus of telethonin, were mutated into serines using standard PCR
(24). A 49er primer
(5'-ATCCATGGCTACCTCAGAGCTGAGCAGCAGCGAGGTGTCGGAGGAGAAC-3') including two
point mutations and the N-terminal cloning site (NcoI) and
the C-terminal cloning primer (5'-AAAGGTACCTTAGCCTCTCTGTGCTTCCTGG-3') were designed for the point mutation of Cys-8 and Cys-15. The PCR
fragment was restricted by NcoI and KpnI and
cloned into the expression vectors pETM11.
Cloning and Heterologous Expression--
All bacterial
transformants with recombinant plasmids were grown in LB medium with
appropriate concentrations of antibiotics. A construct covering the
Z1Z2 N-terminal domains of titin (residues 1-200) was cloned into
pET6d and pETM6d vectors. In pET6d, Z1Z2 directly was fused with a
His6 tag. In pETM6d, there was a TEV proteinase
cleavage site between the His tag and Z1Z2. Ampicillin (100 µg/ml)
was added to the LB medium when Z1Z2 was expressed on either vector.
The full-length (residues 1-167) and an N-terminal truncation
(residues 1-90) of telethonin were cloned onto the pETM11 vector,
leading to constructs pETM-C167 and pETM-C90, respectively. They
contained a kanamycin resistance gene and were expressed in LB medium
supplemented with 50 µg/ml kanamycin. All constructs encoding
telethonin contained a kanamycin resistance gene and were expressed in
LB medium supplemented with 50 µg/ml kanamycin. The overnight
cultures were grown in LB medium at 37 °C, diluted 50-fold, and
grown to an A600 nm of 0.6. The cells
were harvested 4 h after induction with 1 mM IPTG.
Protein Purification and Sample Preparation--
All proteins
were purified using sequential chromatography on
nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen, Hilden, Germany),
MonoQ, and Sepharose 75 gel filtration columns. Z1Z2 was isolated from
the soluble fraction. Harvested cells were resuspended in lysis buffer
(25 mM Tris/HCl, pH 8.0, containing 300 mM
NaCl) supplemented with 1 mg/mg Lysozyme and 0.01 mg/ml DNase I, and were lysed by pulsed sonication (6 min, 30% power, large probe, Fisher
Scientific model 550). Cell debris was removed by centrifugation at
150,000 × g for one hour. The supernatants were
applied to Ni2+-NTA (Qiagen) and equilibrated with
lysis buffer. The bound proteins were eluted with 400 mM
imidazole in lysis buffer. The eluted fraction was dialyzed against 25 mM Tris/HCl, pH 8.0, 1 mM EDTA. The dialysate
was applied to a MonoQ ion exchange column (Amersham Biosciences, HR
16/10) previously equilibrated with 25 mM Tris/HCl, pH 8.0, 1 mM EDTA.
Heterologously expressed telethonin was only found in inclusion bodies.
After lysis and centrifugation, the pellets were dissolved in
denaturing buffer (8 M urea in 25 mM Tris/HCl,
pH 8.0). The solutions were applied to a Ni-NTA column and equilibrated
with the denaturing buffer. The bound proteins were eluted with 400 mM imidazole in denaturing buffer. Z1Z2-telethonin
complexes were formed by adding purified Z1Z2 into telethonin solutions
diluting the solution to 4 M urea concentration. The
protein mixture containing the Z1Z2-telethonine complex was dialyzed
against 6 liters of 0.5 M NaCl in 25 mM
Tris/HCl, pH 8.0, for 2-4 h, then against 25 mM Tris/HCl,
pH 8.0, overnight. The dialysates were applied to a MonoQ ion exchange
column (Amersham Biosciences, HR 16/10) previously equilibrated with 25 mM Tris/HCl, pH 8.0, 1 mM EDTA.
The proteins were released in the same buffer with a gradient of 1-0.5
M NaCl/80 ml at a flow rate of 2 ml/min. The His tag was
cleaved after the Ni2+-NTA column or MonoQ chromatography,
by adding 1/50 tobacco etch virus proteinase and 2 mM
dithiothreitol into the reaction mixture. The reaction was
carried out at room temperature for two hours. Gel filtration was
carried out on a Superose 75 HR10/60 column (Amersham Biosciences)
equilibrated with 25 mM Tris/HCl, pH 8.0, containing 200 mM NaCl. After gel filtration, the protein samples in
buffer (25 mM Tris/HCl, pH 8.0, 200 mM NaCl)
were concentrated to the required value and centrifuged (14,000 rpm,
Microcentrifuge), and the supernatants were used for x-ray solution scattering.
Protein Phosphorylation Analysis--
Phosphorylation of
telethonin and telethonin complexes was carried out essentially as
described (14) using the constitutively active titin kinase mutant
Y170E. Phosphorylation reactions were incubated at 30 °C for 30 min
and analyzed by gel autoradiography and phosphorimaging.
Mass-spectrometric analysis was used to analyze phosphorylation
stoichiometry and sites.
Analytical Ultracentrifugation (AUC)--
Sedimentation velocity
and equilibrium measurements were performed on a Beckman XL-A
analytical ultracentrifuge at 4 °C in 25 mM Tris-HCl, pH
8.0, containing 200 mM NaCl. The sedimentation velocity
experiments were carried out at the and protein concentrations of:
40,000 rpm and 0.86 mg/ml for the full-length telethonin-titin complex
(TE (167)-Z1Z2), 42,000 rpm and 0.83 mg/ml for the truncated telethonin-titin complex (TE (90)-Z1Z2), and 45,000 rpm and 1.3 mg/ml
for the N terminus of titin, with and without polyhistidine tag (Z1Z2,
His-Z1Z2). Scans were taken every 6 min at 280 nm. The sedimentation
equilibrium measurements were performed at three protein concentrations
between 0.36 and 0.90 mg/ml and at four speeds between 16,000 and
29,000 rpm for each sample. Scans were taken after 10 and 12 h of
spinning at every speed at 280 nm. The data were analyzed using DCDT+
(version 1.12) (26) for sedimentation velocity and Ultrascan (version
5.0) for sedimentation equilibrium measurements, respectively.
Ultrascan was a kind gift of Dr. Borries Demeler (University of Texas
Health Science Center, San Antonio, Texas). Partial specific volumes of
0.7316 ml/g (Z1Z2), 0.7297 ml/g (His-Z1Z2), 0.7287 ml/g (TE (90)-Z1Z2),
and 0.7282 ml/g (TE (167)-Z1Z2) and a solvent density of 1.0266 g/ml
were used.
The sedimentation velocity experiments indicated that all four protein
samples were homogenous and the data were fitted to a model for a
single species. The values for the sedimentation (S20,w) and the diffusion
coefficient (D20,w) (27) were
obtained to yield the MM estimates. The sedimentation equilibrium data
were used for direct determination of the MM of the different protein samples.
Scattering Data Collection and Analysis--
The synchrotron
radiation x-ray scattering data were collected on the X33 camera (28,
29) of the EMBL on the storage ring DORIS III of the Deutsches
Elektronen Synchrotron (DESY) using multiwire proportional chambers
with delay line readout (30). The scattering patterns from Z1Z2 and
His-Z1Z2 at protein concentrations between 3 and 80 mg/ml were recorded
at sample-detector distances of 0.7, 1.4, and 3.4 m covering the
range of momentum transfer 0.12 < s < 11.0 nm
Low resolution models of the N terminus of Z1Z2 and of telethonin-Z1Z2
complexes were built using two ab initio methods. The program DAMMIN (22) represents the protein shape as an ensemble of
M
The scattering from the homology model of Z1Z2 (37) was calculated
using the program CRYSOL (38), which takes the scattering from the
excluded volume and from the hydration shell into account. Given the
atomic coordinates, the program fits the experimental scattering curve
by adjusting the excluded volume of the particle and the contrast of
the hydration layer surrounding the particle in solution to minimize
discrepancy (1) between the calculated and the experimental intensities.
Two-hybrid Analysis--
The interaction of titin and full-length
telethonin was monitored by assaying for HIS3-positive phenotype and
Truncation constructs of telethonin were used to obtain a finer mapping
of the titin-binding region by using the constructs Expression and Purification of Z1Z2 and Its Complexes with
Telethonin--
The gene product fragments of titin used in this study
(Fig. 2) were expressed and initially
purified separately. The separate Z1Z2 and His-Z1Z2 constructs were
highly homogeneous as evidenced by gel electrophoresis (Fig.
3) and could be concentrated to about 100 mg/ml. These two constructs yielded an apparent MM around 30 kDa on a
native electrophoresis gel. This value exceeds the calculated MM by
about 50%, suggesting a non-globular shape of the two Z1Z2 constructs.
The expression of the full-length telethonin TE (167) was only
observed in inclusion bodies, whereas the truncated telethonin TE (90)
was partially detected in the soluble fraction. Both telethonin
constructs showed a strong tendency for aggregation and TE (167) was
rapidly degraded so that these constructs were not further purified in
isolated form. The monodisperse in vitro complexes appeared
as single bands in a native electrophoresis gel and could be
concentrated to about 40 mg/ml. The apparent MM were 50-55 kDa and
55-60 kDa for TE (90)-Z1Z2 and TE (167)-Z1Z2, respectively (Fig.
3B).
The complexes of telethonin with Z1Z2 were reconstituted in the
presence of 4 M urea and were subsequently dialyzed against a buffer free from denaturing agents. Initial purification attempts yielded polydisperse samples displaying several MM bands, as evidenced by SDS-PAGE gel electrophoresis gels (data not shown). As the higher MM
bands could result from unspecific disulphide bridge formation by five
cysteines in the telethonin sequence, these cysteines were mutated to
serines. To check whether these mutations might influence the
telethonin folding, the wild type telethonin was treated by reducing
agents (0.2 M dithiothreitol and TCEP). No mobility
difference was observed on SDS-PAGE gel between the wild type and
treated protein. Thiol titration experiments also indicated an absence
of disulphide bridges within the wild type telethonin, and, moreover,
the cysteine to serine mutations did not influence complex formation.
We also assayed their binding in yeast two-hybrid analysis, which again
confirmed that binding to titin was not dependent on the cysteines (not
shown). Furthermore, activity of the in vitro complexes
previously established for a shorter telethonin fragment by a
phosphorylation assay using the constitutively activated titin
kinase mutant Y170E (14) was also carried out for the recombinant
constructs used in this
study.3 Phosphorylation of
Ser-157 was observed for all full-length complexes investigated. No
phosphorylation was observed for the truncated complex in agreement
with the absence of the preference of titin kinase for Ser-157. These
data suggest that Ser-157 is kinase-accessible in the titin complex and
therefore exposed rather than being buried in the heteromultimeric
protein interface. In summary, these data indicated that the in
vitro complexes of mutated telethonin with Z1Z2 constructs were
functionally intact and properly folded.
Determination of Molecular Masses and Complex
Stoichiometry--
The SAXS patterns of all the constructs are
presented on semi-logarithmic plots in Fig.
4 as functions of the momentum transfer. The MM of all the constructs were determined independently by velocity
sedimentation and equilibrium sedimentation in AUC and from the forward
x-ray scattering (Table I). The AUC
sedimentation velocity experiments indicated that all four protein
samples were homogenous, and, hence, the data were fitted to a model
for a single species. In SAXS, the linearity of the initial parts of the scattering patterns in Guinier coordinates (Fig.
5) suggested that the solutions were
monodisperse. These data were complemented by qualitative estimates
from native gel shifts (Fig. 3B), which must, however, be
treated with caution, given the non-globular shapes of the
constructs.
The MM calculated for Z1Z2 and His-Z1Z2 correspond to the expected
values within or close to experimental error. Both equilibrium sedimentation and SAXS data indicate a small increase in MM when the
0.9 kDa polyhistidine tag is added. The experimental data for TE
(90)-Z1Z2 are consistent within experimental error, indicating a MM of
the complex of around 55 kDa. This value does not match the calculated
MM of a 1:1 complex (32.1 kDa). Considering higher associates, a 1:2 TE
(90)-Z1Z2 complex (53.7 kDa) but not a 2:1 TE (90)-Z1Z2 (42.6 kDa) fits
the experimental data. Under the concentration conditions used for SAXS
there is evidence that the full-length complex TE (167)-Z1Z2 forms even
higher associates (see below).
Polyhistidine Tag Localization in the N Terminus of Titin--
The
composite scattering patterns from Z1Z2 and His-Z1Z2 are displayed in
Fig. 4 (curves 1 and 2), and the overall
parameters computed from these curves are presented in Table I. Both
the increase of the Rg by about 0.3 nm and of
Dmax by 1 nm indicate a measurable difference
due to the His tag. The distance distribution functions p(r) computed
from the experimental data (Fig. 6,
curves 1 and 2) are typical for elongated
particles (19). Moreover, the p(r) function of His-Z1Z2 (curve
2) is nearly identical to that of Z1Z2 (curve 1) up to
intraparticle distances of 4 nm and is systematically higher than
curve 1 at larger distances. Given that the His-Z1Z2
contains only seven extra residues compared with non-tagged Z1Z2, these
comparisons unambiguously indicate that the polyhistidine tag is
attached to the extremity of the particle.
The low resolution structures of Z1Z2 and His-Z1Z2 were reconstructed
ab initio using the program DAMMIN from the experimental data up to s = 3.2 nm
As the numbers of residues in the Z1Z2 and His-Z1Z2 constructs are
known (202 and 209, respectively; cf. Fig. 2), their domain structures were also reconstructed by the program GASBOR using the full
scattering patterns up to 0.6 nm resolution. Several DR models of Z1Z2
obtained in independent runs of GASBOR (Fig. 7C) yield a
nearly perfect fit to the experimental data in Fig. 4, curve
1, dashed line. Although the local arrangement of DRs differs from one model to the other, they all display the same overall
appearance with a well defined two-domain structure. The averaged model
(displayed in cyan in Fig. 7D) is similar to that obtained by DAMMIN but reveals the domain arrangement even more clearly. The model of His-Z1Z2 in Fig. 7D (brown)
was obtained by averaging a dozen GASBOR reconstructions providing the
fit to the experimental data in Fig. 4 (curve 2,
dashed line). The superposition of the two averaged models
in Fig. 7D (right column), similar to the DAMMIN
results, permits us to unequivocally identify the polyhistidine tag
region at the upper tip of Z1Z2. Because the tag is attached to
the N terminus of the Z1 IG domain it allows unambiguous assignment of
the Z1 and Z2 IG domains in the low resolution model.
Stoichiometry and Shape of the Truncated Telethonin-Z1Z2
Complex--
The scattering curve of TE (90)-Z1Z2 in Fig. 4
(curve 3) yields the overall parameters in Table I. The
estimated MM significantly exceeds the value expected from the amino
acid sequence of a 1:1 complex (see above). The values of
Dmax and Rg of the TE
(90)-Z1Z2 complex are nearly the same as those of Z1Z2 alone (Table I). The p(r) function in Fig. 6 (curve 3) indicates that the
complex is still an elongated particle but with a larger cross-section than Z1Z2 alone (such a change in p(r) could be caused e.g.
by a side-to-side association of elongated particles).
To obtain a further unbiased estimate of the shape and
stoichiometry of the TE (90)-Z1Z2 complex, its low resolution model was
restored ab initio using the program DAMMIN, which runs
without assumptions about the MM. The independent reconstructions
yielded the fit to the experimental data in Fig. 4 (curve 3,
solid line), and the averaged model is displayed in Fig.
8A as yellow
spheres. The ratio of the excluded volume of the complex (Table I)
to that of Z1Z2 (2.4) is close to the MM ratio of TE (90)-Z1Z2 to Z1Z2 alone (2.5), which further corroborates the view that TE (90)-Z1Z2 is a
1:2 complex. As displayed in Fig. 8A, middle
column, the model of TE (90)-Z1Z2 can accommodate two antiparallel
Z1Z2 models leaving room for telethonin in the middle of the complex.
Assuming that TE (90)-Z1Z2 is a 1:2 complex containing a total of 494 residues, ab initio reconstruction was also performed by
GASBOR yielding the fit to the experimental data in Fig. 4 (curve
3, dashed line). The averaged model displayed in Fig.
6A, right column, is similar to that obtained
from DAMMIN.
Stoichiometry and Shape of the Full-length Telethonin-Z1Z2
Complex--
The scattering pattern of the TE (167)-Z1Z2 complex is
displayed in Fig. 4 (curve 4), and the linearity of the
Guinier plot (Fig. 5) suggests that the solution is monodisperse. The
p(r) function in Fig. 6 is characteristic for a highly elongated
particle, and the estimated MM (95 ± 9 kDa) is nearly twice that
of the truncated TE (90)-Z1Z2 complex. Moreover, the
Dmax and Rg values (Table
I) are also much larger than the corresponding parameters of TE
(90)-Z1Z2. These results suggest that full-length telethonin in complex
with Z1Z2 promotes further dimerization of the 1:2 complexes observed
for TE (90)-Z1Z2. It is interesting that the gel-filtration (Fig.
3B) and AUC sedimentation velocity data (Table I) suggest a
significantly smaller value for the MM, compatible with a 1:2 TE
(167)-Z1Z2 complex. This discrepancy suggests that the association of
this 1:2 complex into higher oligomers under the experimental
SAXS conditions may be transient and concentration-dependent.
The low resolution shape of the TE (167)-Z1Z2 complex was determined
ab initio using DAMMIN to yield the fit to the experimental data in Fig. 4 (curve 4). The excluded volume of the complex
is about twice that of truncated TE (90)-Z1Z2, suggesting a 2:4
stoichiometry. Indeed, the averaged TE (167)-Z1Z2 model in Fig.
8B (yellow beads) can be well represented as an
antiparallel complex of two TE (90)-Z1Z2-like particles. Two models of
TE (90)-Z1Z2 are tentatively positioned in Fig. 8B
(right column) inside the model of TE (167)-Z1Z2 complex. The additional volume in the central part of TE (167)-Z1Z2 may accommodate the additional 154 residues of the two telethonin molecules. However, the low resolution of the models does not permit us
to draw definitive conclusions. DR modeling using GASBOR allowed to
neatly fit the experimental data (Fig. 4, curve 4, solid line) and yielded a virtually identical
average shape at low resolution (not shown).
Accuracy of the Stoichiometry Analysis--
The accuracy of the MM
determination by SAXS is limited, in particular, by the uncertainty in
the measured protein concentrations required for the data
normalization. The estimated MM values alone may thus not be
sufficiently accurate to determine the oligomeric composition and
stoichiometry of a complex. For this reason, we also analyzed the
excluded (Porod) volumes of the particles, taking advantage of the fact
that the Porod volume is readily computed without model assumptions and
does not depend on the normalization of the scattering data. As seen in
Table I, the Porod volumes are in good agreement with the MM estimates
and allow us to reliably determine the stoichiometry of the
telethonin-Z1Z2 complexes. The stoichiometry is further confirmed by
direct comparison of ab initio models of different
constructs in Fig. 8. For three of four constructs (Z1Z2, His-Z1Z2, and
TE (90)-Z1Z2) the MM estimates obtained by SAXS are in excellent
agreement with the AUC results.
The only exception is for the TE (167)-Z1Z2 complex, for which the
results of SAXS and AUC differ by a factor of almost two. This
observation may be due to a concentration-dependent
monomer-dimer equilibrium of a 1:2 TE (167)-Z1Z2 complex in solution.
At low concentrations (<1 mg/ml) used in AUC, the equilibrium is
shifted toward complexes with predicted 1:2 stoichiometry, as observed for the truncated TE (90)-Z1Z2 under all experimental conditions. Conversely, at concentrations above 2 mg/ml, generally employed for
SAXS, the change in stoichiometry of the complex from 1:2 to 2:4 is
consistent with the dimerization of the stable 1:2 complex. Only one TE
(167)-Z1Z2 preparation displayed nearly monomeric behavior up to
concentrations 5 mg/ml in SAXS (Dmax = 15 nm,
MM = 60 kDa), but this result could not be reproduced.
Visualization of a Seven Residues Tag and Reliability of ab initio
Modeling--
Ab initio interpretation of solution
scattering data in terms of three-dimensional models is, generally
speaking, not unique. To reduce the uncertainty, repetitive runs were
performed and analyzed to yield the averaged models containing the
features common to all individual reconstructions. The structural
conclusions are based on these averaged models and different methods
(bead modeling using DAMMIN and DR modeling using GASBOR) yielded very similar results. The independently obtained model of the His-tagged protein has the same overall appearance as that of the native Z1Z2
(Fig. 7) and permits a direct visualization of the tag. To our
knowledge, this is the first time that a tag containing only seven
residues is located using solution scattering. Of course, this
visualization has been greatly facilitated by the fact that it is
attached to the very periphery of a long molecule, but the very fact of
localizing such a small label underlines the improved potential of SAXS
as a structural method. The consistency between the models of Z1Z2 and
its complexes with telethonin (see overlaps in Fig. 8) adds further
credit to the ab initio models.
Using an NMR structure of an IG domain from the I-band of titin (39) a
homology model was predicted for the two N-terminal IG domains of
titin, Z1, and Z2, with a tentative ab initio prediction of
the linker connecting the two IG domains (Ref. 37 and Fig. 7). The
elongated shapes of the two individual domains derived from solution
scattering are compatible with those of the homology model. Instead of
a linear arrangement of Z1-Z2 in the homology model, the solution
scattering models suggest that the two domains are tilted around the
long axes of the molecule (see overlap in Fig. 7B,
rightmost column). Therefore, it is not surprising that the
scattering curve computed from the predicted model (Fig. 4, curve
1, open circles) would fail to fit the experimental
scattering from Z1Z2. It cannot be excluded that the hinge region
between Z1 and Z2 is flexible, allowing the two domains to adopt
different orientations. In this case, a SAXS-derived model would
correspond to an average over the different conformations in solution.
Is Telethonin a Z-disk Cross-linker?--
The SAXS analysis,
supported by AUC and native gel electrophoresis data, indicates that
the stoichiometry of the well documented complex of the N terminus of
the giant muscle protein titin and telethonin differs from the expected
1:1 value (11, 12). All our experimental data are indicative of a 1:2
complex that might further associate into larger assemblies when the C
terminus of telethonin, which is not required for complex formation
(11), is present. Secondary structure prediction programs characterize most of the C terminus of telethonin as a low complexity region. However, at present the structural impact of this part of telethonin on
complex formation and the extent to which it may function autonomously remain unclear. It would be interesting to test whether the C-terminal phosphorylation (14) has an effect on complex association.
The molecular basis of the interaction between the N terminus of titin
and telethonin is a key for understanding the anchoring mechanism of
titin within the Z-disk. Immunolabelling data from a number of
independent studies (11, 12) locate the N terminus of titin and
telethonin on the periphery of the sarcomeric Z-disk. Our data suggest
a cross-linking function for telethonin, connecting two titin molecules
at their N termini. If telethonin acts as a titin-specific
cross-connecting anchor, it remains to be determined how this small
protein is locked into the Z-disk in a regulated fashion that would
allow its selective disappearance (16). Although previous data (10)
revealed how the N terminus of titin cross-links with the other Z-disk
anchored filament system, actin, via staggered
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin and several small proteins
that mostly have been located in the Z-disk by immunofluorescence
microscopy (5, 6). The number of cross-links is controlled by binding
of
-actinin to a number of N-terminal Z-repeats in titin (7), which
are varied by differential splicing in a muscle-specific fashion (8).
This mechanism is regulated by specific Z-disk lipids such as
phosphatidylinositol-bisphosphate (9). The stability of this
cross-linking network is critical for the initialization of
myofibrillogenesis during development and its capacity to resist to
active and passive strain during muscle function (6, 9, 10).
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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. DNA manipulations and
E. coli transformations were carried out using standard
methods (24). The sequences of all cloned PCR products and mutants were
confirmed by sequencing (SEQLAB, Göttingen, Germany).
E. coli BL21(DE3) was used as the expression host. The expression vectors pET6d and pET24d were obtained from Novagen. pETM6d and pETM11 were modified from pET6d and pET24d, respectively, by introducing a tobacco etch virus proteinase
cleavage site between the His tag and cloned gene.
1 (s = 4
sin(
)/
where 2
is the
scattering angle and
= 0.15 nm is the x-ray wavelength). For
the telethonin-Z1Z2 complexes, samples with concentrations between 3 and 20 mg/ml were measured at sample-detector distances of 1.4, 1.8, and 2.4 m covering the range 0.14 nm
1<
s < 5.5 nm
1. The data were normalized to
the intensity of the incident beam, corrected for the detector
response, the scattering of the buffer was subtracted, and the
difference curves were scaled for protein concentration. All procedures
involved statistical error propagation using the program
SAPOKO.2 The low angle data
measured at lower (<10 mg/ml) protein concentrations were extrapolated
to infinite dilution following standard procedures (19) and merged with
the higher angle data to yield the final composite scattering curves.
The maximum dimensions of the particles Dmax
were estimated using the orthogonal expansion program ORTOGNOM (31).
The forward scattering I (0) and the radii of gyration Rg were evaluated using the Guinier
approximation (32) assuming that at very small angles (s < 1.3/Rg) the intensity is represented by
I(s) = I (0) exp(
(sRg)2/3).
These parameters were also computed from the entire scattering patterns
using the indirect transform package GNOM (33), which also provides the
distance distribution functions of the particles p(r). The MM of
the solutes were calculated by comparison with the forward scattering
from reference solutions of bovine serum albumin (MM = 66 kDa).
1 densely packed beads inside a search volume (a sphere of diameter Dmax). Each bead belongs either to the
protein (index = 1) or to the solvent (index = 0), and the
shape is thus described by a binary string of length M. Starting from a
random string, simulated annealing (34) is employed to find a compact
configuration of beads minimizing the discrepancy
between the
experimental Iexp(s) and the
calculated Icalc(s)curves as in
Equation 1,
where N is the number of experimental points,
c is a scaling factor, and
(Eq. 1)
(sj) is
the experimental error at the momentum transfer
sj. The x-ray scattering curves at higher angles
(starting approximately from s = 2.5 nm
1)
contain significant contribution from the internal particle structure,
which must be removed prior to the shape analysis. For this, a constant
given by the slope of an s4I(s) versus s4 plot, is subtracted from the experimental data to ensure
that the intensity would decay as s
4 following Porod's
law (35) for homogeneous particles. The resulting "shape
scattering" curve (i.e. scattering due to the excluded volume of the particle with unit density) in the range up to
s = 3.2 nm
1 was used for ab
initio shape restoration. The excluded volume of the hydrated
particle (Porod volume) was computed from the shape scattering curve
using the following Equation 2 (35).
The outer parts of the scattering patterns (s>3.5
nm
(Eq. 2)
1) dominated by the scattering from the internal
structure were discarded in the shape analysis. In a more versatile
approach implemented in the program GASBOR (23), the protein is
represented as a collection of dummy residues (DR). Starting from
randomly positioned residues, a chain-compatible spatial distribution
of DRs inside the search volume is found by simulated annealing. The DR
method permits fitting data up to 0.5 nm resolution, but the number of residues must be known a priori. In all cases, one or
two dozen DAMMIN and GASBOR reconstructions were performed for each of
the proteins, and the independent models were averaged to yield the most probable low resolution model of the protein. For this, all possible pairs of models were aligned using the program SUBCOMB (36),
and the model giving the smallest average discrepancy with the rest was
taken as a reference. All other models were aligned with the reference
model of a density map of beads, or DRs was computed and cut at a
threshold corresponding to the excluded particle volume.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-galactosidase activity. Activation of both reporter genes was
scored as interaction. Only the construct Z1Z2 was found to interact
with telethonin, whereas the two individual IG domains Z1 and Z2 did
not interact (data not shown). Both single domains Z1 and Z2 were
extended at their C and N termini by 10-12 residues, respectively, to
test the contribution of the linking region between the two IG domains. They behaved as the short single domain constructs in showing no
interaction detectable by the two-hybrid system. This suggests that the
linking region between Z1Z2 does not contribute significantly to
titin/telethonin interaction, or that such an interaction may depend on
secondary structure, or is localized on a non-linear epitope.
C1,
C2 (both
containing the low-complexity, serine-rich C-terminal region), and
N1,
N2, and
N3 (Fig. 1). Growth
on His plates and
-galactosidase activation was observed for
N1
to
N3 but not for the C-terminal fragments
C1 and
C2. We
concluded from these observations that the first 90 residues of
telethonin are sufficient for titin binding, in qualitative agreement
with previous data showing that deletion of residues 4-78 abolished
titin binding (11).
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Fig. 1.
Yeast two-hybrid analysis of telethonine
truncation constructs to monitor interactions with Z1Z2.
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[in a new window]
Fig. 2.
Schematic presentation of the protein
constructs and their acronyms. The residue numbers of the target
sequences are given. Additional residues at the N termini and C termini
are numbered with and +. All cysteines in the wild type
telethonin (Cys-8, Cys-15, Cys-18, Cys-47, and Cys-127; indicated by
C) were mutated to serines in this study.
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Fig. 3.
SDS (A) and native
electrophoresis gels (B) of the purified Z1Z2
(1), His-Z1Z2 (2), TE (90)-Z1Z2
(3), and TE (167)-Z1Z2 (4). The
MM markers are indicated by M.
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[in a new window]
Fig. 4.
X-ray scattering patterns from Z1Z2
(1) His-Z1Z2 (2), TE (90)-Z1Z2
(3), and TE (167)-Z1Z2 (4). The
experimental data are given as dots with error
bars, DAMMIN fits as dashed lines, GASBOR fits as
solid lines. The scattering from the homology model of Z1Z2
(37) is displayed as open circles. The scattering patterns
are displaced by one logarithmic unit for better visualization.
Summary of the experimental and model parameters of Z1Z2 and complexes
with telethonin
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Fig. 5.
Guinier plots of the x-ray scattering
patterns from different Z1Z2 constructs extrapolated to zero solute
concentration. The sequence of samples is as in Fig. 2.
Dots with error bars, experimental data;
straight lines, Guinier fits (note that the Guinier region
is shorter for TE (167)-Z1Z2). The scattering patterns are displaced by
one logarithmic unit for better visualization.
View larger version (22K):
[in a new window]
Fig. 6.
Distance distribution functions of different
Z1Z2 constructs computed from the x-ray scattering patterns using the
program GNOM. The sequence of samples is as in Fig. 2. The p(r)
functions are normalized to unity at their maximum.
1. Fig.
7A displays several models of
Z1Z2 obtained by independent DAMMIN runs (all of them yield the same
fit to the experimental data in Fig. 4, curve 1, solid
line). Despite differences in details, all models have the same
overall elongated shape with two distinct domains. This two-domain
structure is further enhanced in the averaged model (displayed in
cyan in Fig. 7B). The excluded volume of His-Z1Z2
is about 4 nm3 larger than that of non-tagged Z1Z2 (Table
I), which is compatible with the increase in MM due to the His tag. The
shape of His-Z1Z2 is similar to that of Z1Z2, but it displays as extra
mass at the upper extremity of one of the two domains (cf.
the overlap in Fig. 7B).
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Fig. 7.
A, bead models of Z1Z2 obtained ab
initio in five independent DAMMIN (from left to
right). B, averaged DAMMIN models of Z1Z2
(cyan beads) and His Z1Z2 (brown beads) and their
overlap. The rightmost panel displays the overlap of the
ab initio model of Z1Z2 (transparent beads) with
the homology model (37) (red dots). C, dummy
residue models of Z1Z2 obtained ab initio in five
independent GASBOR runs (from left to right).
D, averaged GASBOR models of Z1Z2 (cyan beads)
and His-Z1Z2 (brown beads) and their overlap. In all
panels, the middle and bottom rows are rotated
counter clockwise by 900 around the y and
x axis, respectively.
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Fig. 8.
A, Ab initio low resolution
shape of TE (90)-Z1Z2 obtained by averaging twelve DAMMIN models
(yellow beads, left panel) and this model as
semitransparent beads superimposed with two antiparallel DAMMIN models
of Z1Z2 (cyan and green beads, middle
panel). The right panel displays the model of TE
(90)-Z1Z2 (brown beads) obtained by averaging twelve GASBOR
models. B, the averaged DAMMIN model of TE (167)-Z1Z2
(yellow beads). On the right panel, two DAMMIN
models of TE (90)-Z1Z2 (cyan and green beads) are
tentatively positioned inside the TE (167)-Z1Z2 models. The orientation
of the models is as in Fig. 4.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin crosslinks,
our model suggests telethonin-mediated auto-anchoring of titin dimers
in the Z-disk. To further elucidate the biological significance of
these titin-telethonin interactions this complex needs to be accurately
located and oriented within the Z-disk of myofibrils.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Annalisa Pastore for providing the homology predicted model of Z1Z2.
![]() |
FOOTNOTES |
---|
* 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.
¶ Supported by the Improving Human Potential Program of the European Commission (IHP-RTN2-2001-00451).
** To whom correspondence may be addressed: EMBL c/o DESY, Notkestrasse 85, D-22603 Hamburg. Tel.: 49-40-89902-126; Fax: 49-40-89902-149; E-mail: wilmanns@embl-hamburg.de.
Supported by the International Association for the Promotion of
Cooperation with Scientists from the Independent States of the Former
Soviet Union (Grant 00-243).
¶¶ To whom correspondence may be addressed: EMBL c/o DESY, Notkestrasse 85, D-22603 Hamburg. Tel.: 49-40-89902-125; Fax: 49-40-89902-149; E-mail: svergun@embl-hamburg.de.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M210217200
2 M. H. J. Koch and D. I. Svergun, unpublished data.
3 P. Zou and M. Wilmanns, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
MM, molecular mass;
SAXS, small-angle x-ray scattering;
Ni-NTA, nickel-nitrilotriacetic
acid;
AUC, analytical ultracentrifugation;
DR, dummy residue;
TCEP, Tris(2-carboxyethyl)phosphine;
IPTG, isopropyl-1-thio--D-galactopyranoside;
IG, immunoglobulin;
TE, telethonin.
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