Eur. J. Biochem. 230, 752-759 (1995) 0 FEBS 1995

A calmodulin-binding sequence in the C-terminus of human cardiac titin kinase Mathias GAUTEL', Maria A. CASTIGLIONE MORELLI', Mark PFUHL', Andrea MOTTA' and Annalisa PASTORE ' EMBL, Heidelberg, Germany

' Istituto per la Chimica de Molecole di Interesse Biologico del CNR, Arc0 Felice, Italy

(Received 3 April 1995) - EJB 95 034513

Dipartimento di Chimica, Universith della Basilicata, Potenza, Italy

The giant muscle proteins of the titin family, which are specific for the striated muscles of vertebrates and invertebrates, contain as a common feature a catalytic protein kinase domain of so far unclear function and regulation. In myosin light chain kinase, a family evolutionarily related to titin, kinase regulation is achieved by calmodulin binding to a region of the kinase C-terminus which bears similarity to the sub- strate. A calmodulin-binding sequence has also been identified in the C-terminus of the Aplysia twitchin kinase. In analogy, we identified a putative calmodulin-binding site in the titin kinase C-terminal se- quence. The expressed catalytic domain itself and a series of synthetic peptides from this region were tested for their ability to bind calmodulin. Biochemical data indicate that titin kinase as well as peptides from its C-terminus bind to calmodulin in an equimolar complex in the presence of calcium. The interac- tion of truncated peptides with calmodulin is, however, weaker than that of myosin light chain kinase. Nuclear magnetic resonance studies showed that these peptides have a tendency to adopt a-helical confor- mations in solution. Helicity increases upon binding of calmodulin in a calcium-dependent fashion, as judged by circular dichroism spectra. We, therefore, propose that this calmodulin-binding region of titin could play a regulatory role for the enzyme, the substrate of which still remains to be identified.

Keywords. Muscles ; titin; connectin ; protein kinase.

Recently, a growing family of giant modular proteins spe- cific for the striated muscles of both vertebrates and inverte- brates has been identified and partially sequenced (reviewed by Trinick, 1991). In vertebrates, the giant protein titin has an ap- parent molecular mass of 3 MDa (Maruyama et al., 1984; Kurz- ban and Wang, 1988) and spans the entire length of a half-sar- comere (1 pm; Furst et al., 1988; Whiting et al., 1989). Se- quencing of the titin C-terminus has revealed a protein kinase domain homologous to that present in the myosin light chain kinase (MLCK) family (Labeit et al., 1992). In invertebrates, a similar domain has been found in the nematode mini-titin twitchin (Benian et al., 1989) and the mini-titin of Aplysia (Heierhorst et al., 1994). Indirect evidence has suggested that a protein kinase might also be present in the mini-titins of other invertebrates (Nave et al., 1991 ; Vibert et al., 1993).

Both MLCK and members of the titidtwitchin family share a similar architecture with the kinase domain flanked by patterns of 100-amino-acid modules which belong to the fibronectin type-111 and immunoglobulin superfamilies (Benian et al., 1989 ; Labeit et al., 1992; Heierhorst et al., 1994). Sequence compari- son of MLCK, twitchin and titin has shown that they are closely evolutionarily related (Higgins et al., 1994). Therefore, the mo- lecular sites involved in the regulation of enzymic activity might also be arranged similarly, despite a possible divergence in func- tion and substrate specificity.

In the MLCK family, the catalytic core is linked to C-termi- nal immunoglobulin-like modules by a sequence which func-

Correspondence to A. Pastore, European Molecular Biology Labora- tory, Meyerhofstr. I, D-69012 Heidelberg, Germany

Abbreviations. MLCK, myosin light chain kinase; MLC, myosin light chain ; NOESY, nuclear Overhauser effect two-dimensional spectroscopy; TOCSY, total correlation spectroscopy; EDC, l-ethyl-3- (3-dimethylaminopropy1)carbodiimide.

tions both as autoinhibitory pseudo-substrate for the enzyme and as a binding site for calmodulin (Kennelly et al., 1987; Ikebe et al., 1989; Olson et al., 1990; Bagchi et al., 1992). Binding of calmodulin to its target sequence involves structural changes that release the pseudo-substrate sequence from the active-site cleft of the enzyme and allow the binding of myosin light chains (MLC) as substrates (Knighton et al., 1992; Bagchi et al., 1992). Structural investigation of the twitchin kinase domain demon- strated that the C-terminus of this kinase forms distinct second- ary structures and intimate contacts with the substrate-binding cleft in an autoinhibitory fashion (Hu et al., 1994). For the Aplysia twichin kinase, a high-affinity calmodulin-binding se- quence was identified in the C-terminal autoinhibitory region (Heierhorst et al., 1994).

However, the sequence similarity between MLCKs, twitchin and titin kinase is largely inconclusive in the regulatory C-termi- nal segment of MLCKs (C-terminal to the conserved HPWL box; see Fig. 1). As in MLCK, the titin kinase C-terminal re- gion, which links the catalytic core and the first immunoglobulin domain of the titin C-terminus, shows an abundance of basic and hydrophobic residues, and almost complete absence of negative charges and helix-breaking residues over a sequence of about 20 amino acids with predicted a helical structure. These require- ments are all essential for the interaction with the highly nega- tively charged and hydrophobic surface of calmodulin (Ikura et al., 1992; Meador et al., 1992; Meador et al., 1993). Calmodulin binding could, therefore, be a common feature of the titid twitchin protein kinase family.

Starting from these premises and as part of a long-term pro- ject to characterize the structure and function of titin, we have studied the calmodulin-binding behaviour and the conforma- tional properties of a number of overlapping synthetic peptides

Gautel et al. (Eur J. Biochem. 230) 753

T D M W S L G m 192

315 310 313 309

CaM-binding site of skMLCK

Fig. 1. Multiple sequence alignment of the catalytic domain of rabbit smooth and skeletal MLCK, titin kinase and twitchin kinase. Pairwise similarity was calculated using the UWGCG-package program PILEUP and aligned using PRETTY-PLOT. Conserved residues are boxed and the calmodulin-binding site of rabbit skeletal MLCK is underlined. skMLCK, skeletal myosin light chain kinase ; smMLCK, smooth muscle myosin light chain kinase; titin, titin kinase; twitchin, twitchin kinase.

encompassing the titin kinase C-terminus. As a result, we have identified a stretch of 20 amino acids which bind calmodulin and which is possibly involved in the regulation of the titin kinase.

MATERIALS AND METHODS

Peptide synthesis. Peptides were synthesized by the EMBL peptide service and were shown to be 98% pure by HPLC and mass spectrometry. Peptide sequences were taken from Ikura et al. (1991) for M13 and from the titin kinase sequence (Labeit et al., 1992; Labeit, S., EMBL data library AC X64697), and were:

p7, IRTLKHRRYYHTLIKKDLNMV

VSAARISCGGAIRSQKG ;

P8, HTLIKKDLNMVVSAARISCG ;

p9, KDLNMVVS AARISCGGAIRSQKG ;

p10, STKVIRTLKHRRYYHTLIKKD .

Protein preparations. Calmodulin from human heart was expressed in Escherichiu coli transformed with the calmodulin expression plasmid pCaMl. The human cDNA for calmodulin was isolated from human cDNA by PCR (Saiki et al., 1985) on the basis of the published cDNA sequence of human calmodulin (Wawrzynczak and Perham, 1984; Labeit, S., EMBL data li- brary AC M27319). Using the endogenous NcoI site at the start ATG codon and a BumHI site introduced via the 3' primer, the coding sequence was cloned into the NcoI and BamHI sites of the PET 8c plasmid (Studier et al., 1990). The identity of the cDNA was confirmed by DNA sequencing of the expression plasmid. Transformed E. coli BL21 [DE3] pLysS (Studier et al., 1990) were grown to A,, = 0.8 and induced in a fourfold dilu- tion in Luria-Bertani medium by addition of 0.2 mM isopropyl- P-D-thiogalactoside. Cells from 1 litre culture were harvested after 4 h induction and lysed by sonication with a Branson soni- fier in 30 ml 20 mM potassium phosphate, pH 7, 100 mM NaC1, 5 mM EDTA. Calmodulin was purified from induced cells with a yield of 100 mg/litre culture in modification of the procedure described (Dedman and Kaetzel, 1983). The cell lysate was

centrifuged in a Sorvall SS34 rotor at 15 000 rpm and the soluble supernatant, containing all the expressed calmodulin, was heated to 90°C in a Philips microwave oven for 20 s at maximal setting and immediately chilled in dry-ice-cooled methanol. Denatured E. coli protein was pelleted as described above and the superna- tant adjusted to 20 mM Mes, pH 6, l mM EDTA (buffer A) by dialysis. The dialysate was applied to a 50-ml DEAE-Sephacel column. The column was washed with buffer A and developed with a gradient of 0-500 mM NaCl in 300 ml buffer A. Calmo- dulin eluted in a single peak at the end of the gradient and was 99 % pure as judged by SDSPAGE (Laemmli, 1970), mass spec- trometry and NMR analysis. Human cardiac titin kinase was prepared from Spodopteru frugiperdu ( s f Y ) cells infected with a recombinant baculovirus encoding the catalytic kinase domain of titin including the putative regulatory C-terminus (Labeit, S., EMBL data library AC X64697, bp 7414-8343). The enzyme was purified to homogeneity as will be described elsewhere (Gautel, H., unpublished results). Skeletal MLCK was partially purified from rabbit psoas muscle (Conti and Adelstein, 1991). Mixed MLC were purified from rabbit psoas as described and were depleted of calmodulin by anion-exchange chromatogra- phy (Wagner, 1982). All protein concentrations were determined using a modified Lowry assay (Sigma). Peptide concentrations were determined by ultraviolet spectroscopy as described (Gill and von Hippel, 1989)

Phosphorylation assays. 100 pM calmodulin-depleted mixed MLC were incubated with crude MLCK preparations and 20 nM calmodulin in 200 pl kinase assay buffer (Conti and Adelstein, 1991), containing final concentrations of 100 pM ATP, 1 pCi [y-3zP]ATP (3000 Ci/mmol, Amersham). Calmodulin-binding peptides were added at indicated concentrations. Reactions were incubated at 30°C. At 2-min intervals, 20 p1 aliquots were with- drawn and stopped by filter-precipitation in ice-cold 5 % tri- chloroactic acid as described (Blumenthal et al. 1985). Incorpo- rated radioactivity was measured after addition of 5 ml Ready Safe (Beckman) in a Beckman model LS 8100 liquid scintilla- tion counter. Skeletal muscle MLCK activity was calculated from the initial linear slope of the reactions. Basal activity was

754 Gautel et al. ( E m J. Biochem. 230)

defined as the activity of MLCK in the presence of calmodulin and 5 mM EGTA. Half-maximal inhibition was determined graphically.

Crosslinking experiments. Crosslinking of titin kinase and cal- modulin was performed using a slight modification of the pro- cedure for MLCK (Zot and Puett, 1989) in 20mM Mops, pH 7.0, 100 mM KC1 with 1 mM CaC1, (buffer B) with 5 mM EGTA (buffer C) using the water-soluble zero-length carbodi- imide crosslinker l-ethyl-3-(3-dirnethylaminopropyl)-carbodi- imide (EDC; Sigma). 0.03 nmol titin kinase was incubated at 25°C in 20 pl buffer B or buffer C, with 0.1 nmol calmodulin and 10mM EDC for 30min. Reactions were stopped by addi- tion of 5 p1 4Xsample buffer (Laemmli, 1970) and subjected to electrophoresis on 15 % acrylarnide gels as described (Laemmli, 1970) with a standard molecular-mass marker set (BioRad). Pro- teins were transferred to nitrocellulose membranes as described (Towbin et al., 1979). Titin kinase was detected using a rabbit polyclonal serum directed against the recombinant titin kinase. Calmodulin was detected using a commercial monoclonal anti- body against calmodulin (UBI Upstate Biotechnology). Immu- nocomplexes were visualized by standard techniques using alka- line-phosphatase-conjugated secondary antibodies (Sigma).

Peptide-complex stoichiometry. To determine the stoichiome- try of the complex, a modified version of the gel system de- scribed by Erickson-Viitanen and DeGrado (1987) was used where assays were performed in the absence of urea. 300 pmol calmodulin was incubated with 150, 300 and 600 pmol peptide in 50 mM Tris/HCI, pH 7.5, 0.1 mM CaCl, or 1 mM EGTA. Samples were analyzed on native 15% polyacrylamide gels in the buffer system described by Erickson-Viitanen and DeGrado (1987) in the presence of 0.1 mM CaC1, or 1 mM EGTA.

CD Spectroscopy. CD spectra were recorded on a Jasco 5710 spectropolarimeter that was calibrated with the ammonium salt of 10-(+)-camphorsulfonic acid. The temperature was kept con- stant at T = 20°C using a Neslab RTE-100 water bath. The mea- surements were carried out in a rectangular quartz cuvette from Helma with a pathlength of 1 mm. Sample concentrations were 4 pM for both calmodulin and the peptides in 20 mM Mops, pH 7.5, with 100 mM NaCl and 1 mM CaCI,. A concentration of 2 mM EDTA was used in the control spectra. Spectra were recorded at 200-250 nm with a bandwidth of 2 nm at a speed of 50 ndmin and a resolution of 1 nm. A total of 20 scans were added for each spectrum. Each measurement was repeated three times, then averaged. Analysis, processing and plotting of the spectra were using SNARF (van Hoesel, 1992) and GNUPLOT. Spectra were corrected by subtraction of the baseline and smoothed by fitting to a polynomial. CD spectra containing cal- modulin and/or calmodulin plus peptide were calibrated accord- ing to the concentration of calmodulin alone. CD difference spectra were calibrated according to the concentration of the cor- responding peptide, assuming that the change in helicity is caused by a conformational change in the peptide. Amounts of a-helix were determined using the formula by Chen and Yang (1971).

NMR measurements. The peptides were dissolved in 90% H,O/D,O (by vol.), pH 3.6, or in 80% [SD,],S0/20% H,O (by vol.). The concentrations were in the range 1.1-2.3 mM. The temperature was varied over 17-37°C and the experiments per- formed on a Bruker AM500 spectrometer. 2D total correlation spectroscopy (CLEAN-TOCSY) spectra (Braunschweiler and Ernst, 1983) were acquired with 77 ms and 84 ms mixing times. Nuclear Overhauser two-dimensional effect spectroscopy

a kDa 1 2 3

97-

66-

-+ 45-

b kDa 1 2 3 4

97- 66- -b 45- --+ 32-

32-

21 -

21 - 14-

14-

Fig. 2. EDC-mediated crosslinking and Western-blot analysis fo crosslinked complexes. (a) EDC-mediated crosslinking of expressed titin kinase and calmodulin. SDSPAGE analysis on a 15 % gel of cross- linked complexes visualized by silver staining. Lane 1, marker proteins (BioRad low molecular-mass marker). Lane 2, 1 titin k i n a d 3 calmo- dulin, crosslinked as described in the Materials and Methods in the pres- ence of 5 mM EGTA. Lane 3, crosslinkmg of titin kinase and calmodulin as in described for lane 2 in the presence of 1 mM CaC1,. A crosslinked complex of 55 kDa (arrow) is observed in the presence of calcium ions. (b) Western-blot analysis of crosslinked complexes. Aliquots of the reac- tions in (a) were transferred to nitrocellulose membranes and reacted with anti-(titin kinase) or anti-(calmodulin) IgG. Bound immmunocom- plexes were visualized by alkaline phosphatase colour reactions. Lanes 1 and 2, complexes crosslinked in the presence of EGTA (lane 1) r CaCl, (lane 2), detected by anti-(titin kinase) IgG. Lanes 3 and 4, crosslinked complexes in the presence of EGTA (lane 3) or CaCI, (lane 4) detected by anti-calmodulin IgG as described in Materials and Methods. The 55- kDa complex (indicated by a large arrow) reacts with both the calmo- dulin and titin kinase antibodies. Small arrow, titin kinase; small light arrow, calmodulin.

4

0.4

0.2

0

0.01 0.1 1 10 100 nM peptide

Fig. 3. Inhibition of calmodulin-dependent myosin light chain kinase activity by titin kinase peptides. The activity of skeletal muscle MLCK was assayed in the presence of the titin kinase C-terminal peptides p7, p8, p9 and p10 and the skeletal muscle MLCK peptide M13 at the concentrations indicated. Basal activity was defined as MLCK activity in the presence of 5 mM EGTA under the assay conditions described in the Materials and Methods; 100% activity is the activity in the presence of calmodulin and 1 mM CaCl, and without inhibitory peptides. 0, M13; A, p7; 0, p8; 0, p9; +, p10.

(NOESY) experiments (Macura and Ernst, 1980) were per- formed with different mixing times ranging over 100-300 ms. Spectra were acquired in the phase-sensitive mode with quadra- ture detection in both dimensions using the time-proportional phase-incrementation technique. Processing was carried out on

Gautel et al. (Eur J. Biochem. 230) 755

a Bruker X32 computer using standard software. The datasets acquired in 2KX512 matrices were zero-filled up to 2KX2K using Gaussian and cosine functions in the 0 2 and col dimen- sions, respectively.

RESULTS

Calmodulin binds to the titin kinase and forms a calcium- dependent 1 : 1 complex. SDSPAGE analysis of the crosslinked complex of titin kinase and calmodulin (Fig. 2a) shows that a crosslinked product is obtained in the presence of calcium with an apparent molecular mass of 54 kDa, as judged by the marker proteins, corresponding to the predicted molecular mass of a 1 : 1 complex of titin kinase and calmodulin. In the presence of EGTA, no crosslinked complex is observed, even in the presence of excess calmodulin. The 54-kDa complex reacts with both the titin kinase and the calmodulin antibody (Fig. 2 b), demonstrat- ing that, indeed, a kinase-calmodulin complex is present. We conclude from these experiments that titin kinase contains a sin- gle major site that binds calmodulin in the presence of calcium ions. Several minor bands, immunoreactive to both calmodulin and titin kinase, are also detectable. As their masses do not cor- respond to multiple molecules of calmodulin complexed to titin kinase, and are absent in the presence of EGTA, they are likely to represent conformers of the calmodulin-kinase complex due to multiple internal crosslinking of the polypeptide chains.

Isolated peptides spanning the titin kinase C-terminus bind to calmodulin. Activity assays of skeletal muscle MLCK in the presence of the titin kinase C-terminal peptides p7, p8, p9 and p10 (sequences in Materials and Methods) show that p7, corre- sponding to the entire length of the kinase C-terminus beginning after the HPWL box and linking peptide conserved between the MLCKs and titin kinase, shows the highest affinity for calmo- dulin. This results in competitive inhibition of calmodulin-stim-

1 2 3 4 5 6

Fig. 4. Complex formation of p10 and calmodulin in polyacrylamide gel electrophoresis in the presence of 0.1 mM CaCl,. Lane 1, calmo- dulin control; lane 2, calmodulinlpl0.2: 1 ; lane 3, calmodulin/plO, 1 : 1 ; lane 4, calmodulin/plO, 1 : 2. Lanes 5 and 6, assays from a separate ex- periment in the presence of 1 mM EGTA. Lane 5 , calmodulin; lane 6, calmodulin/plO, 1 :2.

ulated MLCK activity (Figs 1 and 3). Smaller peptides from the titin kinase C-terminal region were designed to map the potential site of interaction with calmodulin to a narrower region. Of these peptides, p10 showed the most pronounced competitive charac- teristics in the inhibition assay. Half-maximal inhibition is achieved at values of 4.2 nM for p7 and 11 nM for p10, while the value for the MLCK calmodulin-binding peptide, M13, is 1.8 nM. These data indicate that calmodulin binding by titin ki- nase peptides is in the nanomolar order, but that their affinities are significantly lower than that reported for M13 (Kd = 1 nM; Blumenthal et al., 1988).

The calmodulin-binding peptide p10 forms a calcium-depen- dent 1 : 1 complex with calmodulin. The peptide p10, the short- est peptide with calmodulin-binding characteristics in the MLCK competition assays, was assayed for its stoichiometry and the calcium-dependence of its complex formation with cal- modulin using a modified version of the band-shift assay de- scribed by Erickson-Viitanen and DeGrado (1987). A single

3

Q2 3

P I a h

7 . 6

7 8

8 0

8 . 2 - s Q

c c

8.4 _" m 0

v - .- E L 0

r I I I I I 8.2 8.0 7.8 1 . 6 8 . 4

Chemical shift (ppm)

Fig. 5. HN/HN connectivities in a NOESY experiment recorded on p7 in Me,SO/H,O 80 % :20 % mixture at 500 MHz and 25°C. The numbers indicate some of the sequential connectivities.

756 Gautel et al. (EUK J. Biochern. 230)

complex band is detected in the presence of calcium, even in the presence of excess p10, indicating that a single binding site is occupied in the complex of calmodulin-peptide. In gels run in the presence of 1 mM EGTA, no band shift is detectable, demonstrating that, under these assay conditions, calmodulin binding by p10 is calcium dependent (Fig. 4).

The peptides have a strong tendency to fold in the helical conformation. NMR spectra in water showed that the peptides are too flexible to allow detection of any specific conformation. The amide resonances are all compressed within 1 ppm and there is no trace of NOE effects typical of helical or other stable secondary structures. As for the titin peptides, M13 has been shown to be unstructured in aqueous solution (Klevit et al., 1985). The following studies were therefore carried out in other more structuring media, such as Me,SOM,O. M13, the peptide encompassing the calmodulin-binding region of MLCK, was studied in the same media for comparison. Virtually complete sequence-specific resonance assignment was achieved for all of the peptides by the standard procedure outlined by Wuthrich (1986). Sequential assignment was obtained by concatenation of HN-HN and Ha-HN NOE connectivities (Fig. 5). Unique resi- dues (such as D17 and, if present, N19, M20 and Q36) and aromatic residues were useful starting points for the assignment. The list of the assignment for p7 is shown (Table 1). The assign- ment of the other three peptides and M13 is also available.

In the Me,SO/H,O mixture, a hydrophilic medium more vis- cous than water, the peptides showed a larger number of NOEs among which several intense HN-HN(i,i+ 1) and HP-HN(i,i+ I ) crosspeaks together with medium-range connectivities Ha- HN(i,i+3) and Ha-HP(i,i+3), indicative of helical regions (Fig. 6). However, many interruptions in the typical helical pattern were present because of severe peak overlap. In particu- lar, NOE effects involving residues V21 and V22 could not be identified because of almost complete degeneracy of their amide resonances in all the peptides which contain this sequence (p7, p8 and p9). The p10 peptide shows a helical character approxi- mately at VO-K15, as identified from NOE patterns, coupling constants and secondary chemical shifts. The helix is much shorter in the p8 peptide, approximately over residue T12-Nl9. For this peptide, the peak overlap was more severe. Neverthe- less, the presence of many typical medium-range NOEs could be definetely ruled out. P9 contains a helical stretch over resi- dues 16- 22 and a more disordered region at residue A24 - Q36. A well resolved long-range NOE from the HN of G30 to the Ha of A24 is indicative of a kink in this region. Finally, p7 contains a helical region at approximately R2-Nl9 and S23-A32. No long-range NOEs were present in this case. In the same medium, M13 was found to be helical at residues R2-A25.

A comparison of these results shows that, apart from border effects due to the tendency of helices to fray at the ends, the titin kinase C-terminus tends to form two distinct helical stretches. The N-terminus titin kinase is consistently longer and better defined.

CD difference spectra indicate a calcium-dependent induc- tion of a helical conformation in the peptides by calmodulin. CD difference spectra recorded in the presence of Ca2+ indicate a strong increase of helicity in p7, p8 and p10 compared to the spectra measured for the free petides (Fig. 7 and Table 2). In contrast, p9 gives almost no extra helicity. For p7, p8 and p10, this extra helicity depends strongly on the presence of Caz+ as indicated by the loss of approximately 50% of the induced helic- ity by the addition of EDTA. P10 experiences the biggest change reaching almost 100% of the a helix, while p7 and P8 contain around 75% of the a helix. The peptide M13 used as a control

Table 1. Chemical shifts of the assigned 'H-NMR resonances of the p7 peptide in Me,SOID,O at 25°C and 500 MHz.

Resi- Chemical shift of due ~

NH CaH VpH CYH C6H others

PPm

1 1 3.62 1.76 1.07 1.42 0.88 2 R 8.71 4.40 1.54 1.67 1.45 3.07 3 T 7.83 4.23 3.96 1 .oo 4 L 8.06 4.07 1.57 1.40 5 K 8.06 4.31 2.71 6 H 8.05 4.50 2.90 3.04 7.12 8.42 7 R 8.22 4.15 1.54 1.60 1.42 3.05 8 R 8.17 4.17 1.44 1.53 1.37 2.99 9 Y 7.88 4.37 2.55 2.80 6.56 6.90

10 Y 8.11 4.37 2.68 2.79 6.60 6.95 11 H 8.32 4.62 2.93 3.05 7.26 8.50 12 T 7.87 4.17 3.95 1.02 13 L 8.02 4.25 1.55 1.40 0.79 0.82 14 I 7.82 4.05 1.71 1.07 0.86 15 K 8.04 4.26 2.73 16 K 7.68 4.15 1.67 1.47 1.27 2.75 17 D 8.22 4.48 2.53 2.72 18 L 7.94 4.13 1.68 1.43 0.78 0.83 19 N 8.08 4.46 2.45 2.56 6.88 7.42 20 M 7.83 4.32 1.75 1.90 2.37 21 V 7.82 4.03 1.95 0.80 22 V 7.82 4.06 1.95 0.80 23 S 7.89 4.25 3.53 3.61 24 A 8.08 4.17 1.20 25 A 7.98 4.12 1.18 26 R 7.92 4.18 1.55 1.68 1.45 3.04 27 I 7.70 4.17 1.67 1.03 1.47 0.78 28 S 8.03 4.33 3.60 29 C 8.33 4.59 2.85 3.15 30 G 8.27 3.75 31 G 8.05 3.74 32 A 7.98 4.28 1.18 33 I 7.85 4.05 1.68 1.00 1.35 0.75 34 R 8.04 4.13 1.48 1.67 1.27 35 S 7.94 4.27 3.54 3.62 36 Q 8.14 4.19 1.76 1.92 2.12 6.79 7.36 37 K 8.00 4.17 1.67 1.29 1.50 2.73 38 G 8.12 3.75

0.75 0.80

gave values identical within the error margins to previously pub- lished ones (Klevit et al., 1985). Its behavior in the absence of calcium is comparable to that of the titin peptides.

DISCUSSION

In the present work, we have shown that expressed titin ki- nase bearing the putative regulatory C-terminal sequence can be crosslinked to calmodulin at a 1 : 1 ratio in a calcium-dependent manner. A similar behaviour has been previously described for MLCK (Zot and Puett, 1989), although the amount of complex crosslinked for this enzyme was much greater. By using the partly overlapping sub-peptides, we localized the calmodulin- binding motif of titin kinase to within the first 20 amino acids of p7. NMR spectra of synthetic fragments of the titin kinase C- terminus show that they are very flexible in water, in agreement with what has been reported for most of the other calmodulin- binding peptides. For structural investigation, we preferred to use Me,SO/H,O mixtures rather than the more widely used trifluoroethanol to study the conformational tendencies of

Gautel et al. (Eul: J. Biochern. 230) 757

Ha-NH (1,1+2)

Ha-NH (1,1+3) - m

Ha-NH (1.14) - 1 5 10 15 10 n s o u

I R T L K H R R Y Y H T L I K K D L N M V V S A A R I S C G G A I R S Q K G

C 16 21 2% 31 S

K D L N M V V S A A R I S C G G A I R S Q K G

NKNH(i,l+l) " * - * - -

I

Ha-NH (1,14) - 16 11 2% 31 la

K D L N M V V S A A R I S C G G A I R S Q K G

11 18 n I

H T L I K K D L N M V V S A A R I S C G

B

Htl-NH (l,l+l) =* m

Ha-NH (1,1+2)

(3 38 01 lo

H T L I K K D L N M V V S A A R I S C G

-3 3 5 10 15 D S T K V I R T L K H R R Y Y H T L I K K D

Ha-NH (l,l+i) * * t t =

Ha-NH (1,1+2)

-3 1 5 10 15

S T K V I R T L K H R R Y Y H T L I K K O

Fig.6. A survey of the most significant classes of observed distances for the four peptides studied. (A-D) refer to p7, p8, p9 and p10, respectively. Sequential connectivities are classified into categories according to their intensities and indicated by the height of the bars. Filled circles indicate J,,.,,. coupling constants estimated to be smaller than 5 Hz. Asterisks mark overlapping peaks.

these peptides. Although trifluoroethanol acid is a highly helical- inducing solvent, it has been shown that it can induce helical conformation non-specifically even in stretches which assume different secondary structures in the native context (Soennichsen et al., 1992). Me,SO/D,O is, in contrast, a highly polar medium which increases solvent viscosity and has been shown to stabi- lize a structure by slowing down the molecular motions (Shich- man and Amey, 1971 ; Amodeo et al., 1991). In this medium, all the peptides show helical behaviour, although differentially along the sequence. The N-terminal half of p7 has the strongest tendency to form helices. In contrast, p9, which spans the p7 C- terminus, contains the shortest helical stretch. This tendency of the N-terminal region of p7 is stabilized by calmodulin in the

complex, as demonstrated by CD difference experiments. Inter- estingly, the comparison of secondary structure elements from our NMR data with those of the Caenorhabditis elegans twitchin kinase structure indicates a structural homology of their autoin- hibitory C-termini, despite a negligible sequence homology be- tween the titin and twitchin kinase C-termini (Fig. 8). Further- more, a calmodulin-binding region was proposed to be localized in the corresponding region of Aplysia twitchin kinase (Heier- horst et al., 1994).

The affinity of titin kinase for calmodulin, as deduced from the assays with p7 and p10, is significantly lower than that re- ported for M13 (Kd 1 nM; Blumenthal et al., 1985; Blumenthal et al., 1988). We can, therefore, not exclude that, in vivo, the

758 Gautel et al. (Eu,: J. Biochem. 230)

B

200 210 220 230 240 250 200 210 220 230 240 250 h i nm h I nm

Fig.7. CD difference spectra of p9 and p10. (A) Difference spectra for p10 with (bottom) and without (middle) CaZf compared to the spectrum of the free peptide (top). (B) Difference spectra for p9 with (middle) and without (top) Ca2+ compared to the spectrum of the free peptide (bottom). Error bars are displayed at the typical minima of an a helix at 208 nm and 222 nm.

Table 2. Molar ellipticity and amount of a helix for calmodulin and peptides. Minima in molar ellipticity and a helix contents are shown for calmodulin and all four peptides, together with the control peptide M13. CD spectra recorded for p7 in trifluoroethanol gave a molar ellipticity at 222 nm of -9100 2 700 deg cm2 dmol-', resulting in a content of 22% a helix.

Peptide Calmodulin Ca2+ [@I2, a helix

P7 P7 P7

P8 P8 P8

P9 P9 P9

deg cm2 dmol-' %

- 2400 2 800 -26 000 ? 1800 -12 100 ? 1400

- 3 100 2 800 -25 400 2 2000 -12 050 L 1900

- 4 5 0 0 2 900 - 5 500 IT 2100 - 5 800 2 2000

0 76 32

3 75 31

7 10 11

PI0 - + - 3600 L 800 4 + + -31 800 t 2100 97

PI0 + - - 15 500 L 2200 43 PI0

M13 - + - 1 6 0 0 2 600 0 M13 + + -32 300 ? 2000 99 M13 + - -14 600 IT 1900 41

Calmodulin + + - 1 5 2 0 0 t 800 42 Calmodulin + - -139002 500 38

amphiphilic helical titin kinase C-terminus might bind regula- tory proteins other than calmodulin, or is dislodged from the substrate-binding cleft by other mechanisms. Its affinity, though, is still in the range of other calmodulin-activated enzymes (as compiled by O'Neil and DeGrado, 1990) and allows calcium- dependent calmodulin binding under physiological conditions. However, the presence of calmodulin does not suffice to activate the catalytic kinase activity of our expressed titin kinase directed against unspecific kinase substrates such as dephosphorylated casein, histones or myelin basic protein (data not shown). This may be related to further regulatory mechanisms needed for ki- nase activation, e.g. phosphate turnover on the activation loop, or a high substrate specificity of the enzyme.

Evidence has emerged from DNA sequencing (Benian et al., 1989; Labeit et al., 1992; Heierhorst et al., 1994) as well as

Titin

a-helix

p-strand

Twltchin

Fig. 8. Schematic comparison of secondary structure elements in the regulatory C-termini of titin and twitchin kinase. The C-terminal re- gions of titin and twitchin kinase are shown, with the regions of helical secondary structure experimentally determined by NMR (for titin kinase) and crystallographically (for twitchin kinase, adapted from Hu et al., 1994) boxed.

immunological studies with anti-kinase-directed IgG (Nave et al., 1991; Vibert et al., 1993) that the presence of a catalytic kinase domain is a common feature of the diverse titidtwitchin family of giant muscle proteins. However, the regulation and function of these kinases have been hard to establish so far. Se- quence comparison and evolutionary analysis of titin, twitchin and MLCKs have suggested that these protein families have evolved from a common ancestor most closely related to the MLCK of Dictyostelium (Higgins et al., 1994). This kinase is not regulated by calmodulin and contains no immunoglobulin- like or fibronectin type-I11 domains as compared to the MLCKs in higher organisms (Tan and Spudich, 1990; Tan and Spudich, 1991) or the titidtwitchin kinases. The titin and MLC kinase families, therefore, seem to have evolved their regulatory sites independently and possibly with divergent functions during the evolution of cross-striated muscle (Labeit et al., 1992; Higgins et al., 1994).

Further characterization of the expressed titin kinase is needed to elucidate its biological function. The question of cal- modulin activation of titin kinase in contraction regulation, as for MLCKs, or in other regulatory mechanisms similar to calmo- dulin-kinase 11, must remain open until its substrates and up- stream regulators are found.

We are grateful to R. Jacob for peptide synthesis, V. Adam and S. O'Loughlin for oligonucleotide synthesis, A. M. Voie and H. Erfle for DNA sequencing, M. Mann for mass-spectrometry measurements and P. Brownlie for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschu~ (MG) and the Human Frontier Science Program Organization (AM and AP).

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