Solution Scattering Suggests Cross-linking Function of Telethonin in the Complex with Titin*

Peijian ZouDagger , Mathias Gautel§, Arie Geerlof||, Matthias WilmannsDagger **, Michel H. J. KochDagger DaggerDagger, and Dmitri I. SvergunDagger DaggerDagger§§¶¶

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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).

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains and Plasmids-- All plasmids were constructed in Escherichia coli Strain DH5alpha . 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.

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-1 (s = 4pi sin(theta )/lambda where 2theta is the scattering angle and lambda  = 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).

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 M1 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 chi  between the experimental Iexp(s) and the calculated Icalc(s)curves as in Equation 1,
&khgr;<SUP>2</SUP>=<FR><NU>1</NU><DE>N−1</DE></FR> <LIM><OP>∑</OP><LL>j</LL></LIM><FENCE><FR><NU>I<SUB>exp</SUB>(s<SUB>j</SUB>)−cI<SUB>calc</SUB>(s<SUB>j</SUB>)</NU><DE>&sfgr;(s<SUB>j</SUB>)</DE></FR></FENCE><SUP>2</SUP> (Eq. 1)
where N is the number of experimental points, c is a scaling factor, and sigma (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).
V=2&pgr;<SUP>2</SUP> I(<UP>0</UP>)/<LIM><OP>∫</OP><LL>0</LL><UL>∞</UL></LIM>s<SUP>2</SUP> I(s)ds (Eq. 2)
The outer parts of the scattering patterns (s>3.5 nm-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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Two-hybrid Analysis-- The interaction of titin and full-length telethonin was monitored by assaying for HIS3-positive phenotype and beta -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.

Truncation constructs of telethonin were used to obtain a finer mapping of the titin-binding region by using the constructs Delta C1, Delta C2 (both containing the low-complexity, serine-rich C-terminal region), and Delta N1, Delta N2, and Delta N3 (Fig. 1). Growth on His plates and beta -galactosidase activation was observed for Delta N1 to Delta N3 but not for the C-terminal fragments Delta C1 and Delta 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.

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).


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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.

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.


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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.

                              
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Table 1
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.

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.


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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.

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-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.

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.


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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.

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).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.

Dagger Dagger 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.

    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-beta -D-galactopyranoside; IG, immunoglobulin; TE, telethonin.

    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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