©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Regulatory Domain of Gizzard Calponin
INTERACTIONS OF THE 145-163 REGION WITH F-ACTIN, CALCIUM-BINDING PROTEINS, AND TROPOMYOSIN (*)

Mohamed Mezgueldi (§) , Christiane Mendre , Bernard Calas , Ridha Kassab , Abdellatif Fattoum (¶)

From the (1) Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U 249, Université de Montpellier I, Route de Mende, BP 5051, 34033 Montpellier Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Earlier, we proposed that the interaction of gizzard calponin with F-actin, promoting the inhibition of the actomyosin ATPase activity, involves the NH-termi-nal portion of the calponin segment Ala-Tyr(Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). In this work, we have directly probed this region for actin binding sites using five peptide analogs covering different stretches of the sequence Thr-Ile. Co-sedimentation with F-actin, actomyosin ATPase measurements, and zero-length cross-linking reactions demonstrated that the 19-residue sequence Ala-Ileis essential for actin interaction and ATPase inhibition. Furthermore, each peptide was tested for binding to the Ca-dependent proteins, caltropin and calmodulin, in both an actomyosin ATPase assay and an affinity chromatographic assay. The results revealed the 11-residue segment Gln-Ile, representing the COOH-terminal moiety of the F-actin binding sequence, as a crucial region for the high affinity binding of these regulatory proteins with concomitant removal of the ATPase inhibition. The 153-163 stretch contained also interactive sites for tropomyosin as assessed by affinity chromatography and spectrofluorometry. Collectively, the data support our initial results and highlight the ability of the multifunctional 145-163 region to serve as a potent regulatory domain of the smooth muscle calponin.


INTRODUCTION

Smooth muscle calponin is an actin- and tropomyosin-binding protein which may modulate at the thin filament level the actin-myosin interactions and force generation (1, 2, 3) . Solution studies and in vitro motility assays have shown that its binding to F-actin or F-actin-tropomyosin promotes the inhibition of the actin-stimulated myosin ATPase (4, 5, 6, 7) and actin sliding over myosin (8, 9) . This inhibitory activity can be reversed by complexation of calponin to a calcium-dependent protein, like calmodulin, caltropin, or protein S100b (10, 11) as well as by its site-specific phosphorylation by protein kinase C or Ca-calmodulin-dependent protein kinase II (4, 12, 13) . The identification of a calponin phosphatase has suggested that a cycle of phosphorylation-dephosphorylation of the protein may regulate the cross-bridge activity in vivo (4, 14, 15, 16) . The recently described occurrence of calponin in the actomyosin domain of the smooth muscle cell further strengthens its proposed involvement in the regulation of smooth muscle contraction (17) .

To define the molecular basis of calponin functions, we have earlier investigated the distribution of its regulatory F-actin and calmodulin interactive sites over three chymotryptic fragments isolated from gizzard calponin and accounting for its entire primary structure (18) . We found that all the known biological activities of the protein were associated with its NH-terminal segment spanned by residues 7-182 whereas no detectable function was displayed by the remaining COOH-terminal 183-292 region. Essentially similar observations were subsequently reported by others (13) . Most importantly, our studies, using proteolytic digestions and zero-length cross-linking of the F-actin-calponin complex, strongly suggested but did not prove that the inhibitory binding of actin takes place on the 38-residue stretch between Alaand Tyr, the most exposed and protease-sensitive region on the surface of calponin (3) . Residues present toward the amino-terminal end of this sequence have been proposed to interact with actin. Our data also led to the conclusion that the segment spanning amino acids 61-182 includes major determinants for the recognition of F-actin, calmodulin, and tropomyosin. More recently, the binding stoichiometry for the complex of calponin and calmodulin or caltropin was determined to be 1:2 with the interaction of either calcium-dependent protein at a low and at a high affinity site on the calponin molecule (10, 11) . The existence of the two different sites was confirmed by briefly described titration analyses of the binding of the former proteins to a recombinant fragment of calponin spanning residues 1-228 (19) ; these data further indicated that the weaker binding site was within the NH-terminal segment 2-51 and the stronger binding site was residing between residues 52 and 228, a region which also comprises our putative inhibitory F-actin interactive domain. However, the functional and structural relationships between the latter crucial site and the pair of sites involved in the recognition of the calcium-dependent proteins remain to be elucidated. In the present work, we have assessed the functional coupling between the actin site and the site of tight binding of the calcium-dependent proteins, by determining more precisely the relative location of their respective critical determinants within the covalent structure of calponin. Taking advantage of our recent findings with caldesmon revealing the juxtaposition of the regulatory F-actin and calmodulin binding sequences on the primary structure of its COOH-terminal domain (20) , we have synthesized a series of oligopeptides corresponding to different stretches of the calponin amino acid sequence from Thrto Ile. This segment encompasses the NH-terminal moiety of our proposed F-actin binding region of residues 145-182 (18) . We have probed in detail their biological properties using F-actin co-sedimentation, actomyosin-S-1 ATPase assays, cross-linking reactions with F-actin or calmodulin, and affinity chromatography over immobilized calmodulin or tropomyosin. The data demonstrate for the first time the direct interaction of the 19-residue segment Ala-Ilewith F-actin, Ca-calmodulin, Ca-caltropin, and tropomyosin. Its complexation to actin occurs competitively with the binding of the functional calponin fragment of residues 7-182 and causes the inhibition of ATPase activation in a calmodulin- or caltropin-regulated manner, whereas its association with tropomyosin was without consequences. The interaction of the calcium-dependent proteins and tropomyosin was restricted to the COOH-terminal portion Gln-Ile; in contrast, actin recognition required the entire 145-163 segment. These studies highlight several interaction properties of calponin and provide the experimental evidence for the previously assigned location of its inhibitory actin binding site (18) . They also suggest that the multifunctional features of the 145-163 sequence combined with its close proximity in calponin primary structure to the phosphorylatable residues, Ser/Thr, are likely reflecting the ability of this particular region to act as a potent regulatory domain of the native protein.


EXPERIMENTAL PROCEDURES

Materials

-Chymotrypsin was purchased from Worthington Biochemical Corp. Calmodulin-Sepharose and CNBr-activated Sepharose 4B were from Pharmacia Biotech Inc. The latter material was coupled to tropomyosin according to the manufacturer's specifications. Fmoc amino acids were obtained from Milligen (Millipore). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)() and N-hydroxysuccinimide (NHS) were from Sigma and Serva, Heidelberg, respectively.

Proteins and Peptides

Calponin and tropomyosin from fresh turkey gizzards, F-actin, and chymotryptic myosin subfragment-1 (S-1) from rabbit skeletal muscle were prepared as described previously (18) . Bovine brain calmodulin and chicken gizzard caltropin were purified by published procedures (20, 21) . Tropomyosin was labeled at Cyswith pyrene- N-maleimide as reported earlier (22) . The 22-kDa and 13-kDa NH-terminal chymotryptic fragments of calponin (CH22 and CH13N, respectively) were produced according to Mezgueldi et al. (18) and were purified over immobilized tropomyosin. Synthetic peptides were prepared and purified as reported earlier (20, 23) . They were subjected to amino acid analysis, sequence determination, and molecular mass measurement using electrospray ionization mass spectrometry.

Protein Concentrations

The concentrations of S-1, F-actin, tropomyosin, calmodulin, caltropin, and calponin were measured spectrophotometrically using

On-line formulae not verified for accuracy

= 7.5, 11.7, 2.2, 2.0, 6.8, and 7.0 (18, 21) , respectively. The concentration of the chymotryptic or synthetic peptides was determined from their absorbance at 280 nm and calculated on the basis of their tryptophan content (24) . The concentrations obtained were in good agreement with those determined from the amino acid composition. The concentrations of TY12, AF8, and QI11 were estimated by amino acid analysis.

Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis was carried out in 3-18% or 7.5-20% gradient acrylamide gels (25) . The running buffer was 50 m M Tris, 100 m M boric acid, pH 8.0. Marker peptides used were myoglobin CNBr fragments: 2.6 kDa, 6.4 kDa, 14.6 kDa, and 17.2 kDa. The immunoblotting with polyclonal actin antibodies was performed as reported earlier (18) .

F-actin Binding Assays

The interaction of F-actin and peptides was analyzed by co-sedimentation in 10 m M imidazole-HCl, 30 m M NaCl, 2 m M MgCl, 1 m M NaN, pH 7.0, using a Beckman Airfuge at 140,000 g, for 30 min at 25 °C. The pellets were homogenized in the same buffer and analyzed either qualitatively by gel electrophoresis or quantitatively by HPLC on a reverse-phase Aquapore C-8 column (4 mm 250 mm) eluted with 0-60% acetonitrile gradient containing 0.1% trifluoroacetic acid (20) .

Chymotryptic Digestions

Calponin or its 22-kDa fragment was treated with chymotrypsin at 25 °C using a protease to substrate weight ratio of 1:100, in 10 m M imidazole HCl, 30 m M NaCl, 2 m M MgCl, 1 m M NaN, pH 7.0. At the desired time intervals (0-60 min), protein aliquots were analyzed by gel electrophoresis and assayed for inhibition of the acto-S-1 ATPase after the addition of 2.5 m M phenylmethylsulfonyl fluoride.

Cross-linking Reactions

TW31 (0.15 mg/ml) in 10 m M imidazole HCl, 30 m M NaCl, 2 m M MgCl, 1 m M NaN, pH. 7.0, was mixed with F-actin (0.21 mg/ml) (TW31:actin molar ratio = 8:1) in the presence of 2 m M EDC and 5 m M NHS. The reaction was carried out at 25 °C for 0-30 min in the absence or presence of Ca-calmodulin (5.5 mg/ml) (TW31:Ca-calmodulin molar ratio = 1:8) and was monitored by SDS-gel electrophoresis.

Calmodulin and Tropomyosin Binding Measurements

The binding of peptides to calmodulin was assayed by calmodulin-Sepharose affinity chromatography as described previously (20) .

Affinity chromatography of the peptides over tropomyosin-Sepharose was performed in 10 m M Tris-HCl, 10 m M KCl, 1 m M MgCl, 0.1 m M dithiothreitol, and 1 m M NaN, pH 7.0. Bound peptides were eluted with the same buffer containing 200 m M KCl.

Fluorescence emission spectra of the calmodulin-TW31 complex and of the complexes between pyrene-tropomyosin and synthetic peptides were recorded as reported earlier (20, 22) .

ATPase Measurements

The Mg-ATPase of the skeletal acto-S-1 was determined in a medium (1 ml) containing 50 m M Tris-HCl, 5 m M ATP, 10 m M KCl, 2.5 m M MgCl, pH 7.5, using 0.42 mg/ml F-actin and 0.050 mg/ml S-1. Pwas determined colorimetrically as described previously (18) .


RESULTS

Effect of the Limited Chymotryptic Digestion of the Calponin Fragment of Residues 7-182 on Its Inhibitory Activity-When the isolated 22-kDa NH-terminal chymotryptic fragment of calponin spanning amino acids 7-182 was further incubated with chymotrypsin at a protease to substrate weight ratio of 1:100, it was rapidly and quantitatively converted into the stable peptide designated CH13N (Fig. 1 A). Earlier, this component was shown to derive from the cleavage, under identical conditions, of the intact calponin at Tyr-Alaand was identified as the NH-terminal segment of residues 7-144 (18, 26) . Thus, the direct proteolysis of the 22-kDa fragment did result only in the excision of its COOH-terminal 38-residue stretch, between Alaand Tyr, thought to contain the inhibitory actin-binding region (18) . Most interestingly, when the whole digest of the 22-kDa peptide was subjected in parallel to acto-S-1 ATPase assays during the entire course of the proteolytic process, no change in the inhibitory capability of the peptide was noticed even after a 60-min reaction (Fig. 1 B). The digestion, under the same experimental conditions, of the parent calponin, used as control, also failed to alter significantly the extent of ATPase inhibition. The initial 70% inhibition of the activity tended to level off to the same value (60%) as that found for the digested 22-kDa fragment (Fig. 1 B). These observations strongly suggest that the proteolysis had no deleterious effect on the major structural determinants for specific binding to F-actin that are still operant in the released 38-residue peptide or in its chymotryptic subfragments. There is no doubt that only the presence of the latter fragment(s) in the digest solution is responsible for the observed maintenance of the ATPase inhibition since, as indicated in Fig. 1 C, only the intact 22-kDa fragment but not its isolated 13-kDa NH-terminal portion could inhibit the acto-S-1 ATPase in a concentration-dependent manner. The inefficiency of the 13-kDa fragment was not caused by its isolation since, earlier, we have shown that this material did not co-sediment with F-actin directly added to the total digest of calponin (18) . Also, all of the 13-kDa peptide remained in the supernatant upon centrifugation of a mixture of F-actin and the 60-min digest of the 22-kDa peptide (data not shown).


Figure 1: Chymotryptic digestion of the CH22 fragment and its consequence on the acto-S-1 ATPase inhibition by the fragment. A, CH22 fragment (2.0 mg/ml) in 10 m M imidazole HCl, 30 m M NaCl, 2 m M MgCl, 1 m M NaN, pH 7.0, was digested with -chymotrypsin at 25 °C (protease to substrate weight ratio = 1:100). At the times indicated, the digest was analyzed by electrophoresis on 7.5-20% gradient acrylamide gel. B, at the indicated time intervals, aliquots of the digest of CH22 () were subjected to acto-S-1 ATPase assays. Aliquots of a chymotryptic digest of calponin (), produced under the conditions reported in A, were similarly tested for inhibition of the acto-S-1 ATPase activity (actin:calponin or CH22 molar ratio = 1:0.5). C, acto-S-1 ATPase assays were carried out using increasing concentrations of the isolated fragments CH22 () or CH13N (). 100% ATPase = 0.450 µmol of P/min/mg.



The apparent conservation of the inhibitory function in the proteolytically detached 145-182 region encouraged us to prepare various synthetic peptides covering the NH-terminal sequence 145-163 as probes of the interaction between actin and this region.

Ca-Calmodulin-regulated Interaction of F-actin with the Synthetic Peptides Including the Sequence Ala-Ile

The peptides we have synthesized corresponded to different stretches of the main sequence Thr-Ile. In some peptides, Ilewas replaced by a tryptophan residue. This amino acid conferred characteristic spectral features without influencing the biological activities of the peptide as verified by comparing the functional properties of the peptide analogs comprising either isoleucine or tryptophan at position 163. The synthetic peptides used in this study were designated TW31 (spanning the 31-residue sequence Thr-Trp), TY12 (spanning the 12-residue sequence Thr-Tyr), AW19 (spanning the 19-residue sequence Ala-Trp), AF8 (spanning the 8-residue sequence Ala-Phe), and QI11 (spanning the 11-residue sequence Gln-Ile).

Each peptide represented a single major component showing less than 2% impurity as assessed by reversed-phase HPLC and mass spectrometry (Fig. 2). Amino acid composition analyses, sequence determinations, and molecular masses were consistent with the expected sequences. All peptides were readily soluble in aqueous buffer. We investigated comparatively their binding to F-actin by co-sedimentation as well as by EDC cross-linking in the absence or presence of Ca-calmodulin. We also tested their ability to inhibit the acto-S-1 ATPase and the influence of the Ca-dependent proteins, caltropin and calmodulin, on their inhibitory activity.

Following centrifugation of each peptide in the absence or presence of F-actin (actin:peptide molar ratio, 1:8), the corresponding pellets and supernatants were analyzed either by electrophoresis on a 7.5-20% gradient acrylamide gel or by reverse-phase HPLC. As illustrated in Fig. 3, TW31 and AW19 displayed significant binding to F-actin, and no pelleting of either peptide occurred without actin. The peptide analogs including isoleucine instead of tryptophan at the COOH terminus behaved essentially similarly (data not shown). Because TY12 (Fig. 3) as well as AF8 and QI11 were small-sized peptides, their respective pellet and supernatant fractions could not be analyzed by gel electrophoresis and were subjected to reverse-phase HPLC. No trace of either peptide was found associated with F-actin (data not shown). The actin-binding peptides TW31 and AW19 share a common sequence, spanned by Ala-Ile(Trp), which must include the critical determinants for F-actin binding; this proposal is consistent with the absence of F-actin binding to the difference peptide TY12. On the other hand, the lack of actin interaction with its separated NH-terminal and COOH-terminal moieties, represented by AF8 and QI11, respectively, strongly suggests that the structural integrity of the full-length 19-residue sequence between Alaand Ileis required for F-actin recognition.


Figure 3: Interaction between F-actin and the synthetic peptides. The binding of the synthetic peptides, TW31, TY12, and AW19, to F-actin (actin:peptide molar ratio = 1:8) was analyzed by co-sedimentation at 140,000 g for 30 min at 25 °C followed by electrophoresis of the supernatant and pellet fractions on a 7.5-20% gradient acrylamide gel. The binding of calponin ( CaP) and CH22 to F-actin was assayed similarly as a control. Lanes a and b, supernatant and pellet, respectively, of each peptide alone. Lanes c and d, supernatant and pellet, respectively, of each peptide centrifuged with F-actin.



To assess that the latter critical sequence also significantly contributes to the complexation of the functional 22-kDa chymotryptic fragment of calponin (residues 7-182) to F-actin, we investigated the competition between this fragment and TW31 for binding to F-actin. Fig. 4, A and B, shows the potency of TW31 to displace the proteolytic fragment from actin in a concentration-dependent manner. About 60% of the bound fragment was dissociated from actin at a TW31:CH22 molar ratio near 10. This finding indicates that TW31 competes with CH22 for a common binding site on F-actin. Previously, we have shown that native calponin or CH22 could be specifically cross-linked to F-actin by EDC, and we identified the cross-linked site on the actin carboxyl-terminal region between residues 326 and 355 (18) . The actin binding activity observed for the synthetic TW31 led us to analyze its EDC cross-linking to F-actin. The time course of the reaction is depicted in Fig. 5 A, lane c, using a molar ratio of actin to peptide of 1:8. A new major species of 45 kDa, migrating just above the actin monomer, was generated and its yield increased with the reaction time. It was immunostained with the actin antibodies (Fig. 5 B). The 45-kDa derivative likely represents a 1:1 molar covalent complex of actin and TW31. Its production strongly suggests that the zero-length cross-linking of either calponin or CH22 to F-actin reported earlier (18) was mediated by the active sequence Ala-Ile. Significantly, Fig. 5 A, lane g, shows that the formation of the 45-kDa adduct is almost completely suppressed in the presence of Ca-calmodulin added at an 8-fold molar excess over the synthetic peptide. Instead, a new entity of 21 kDa, with a mobility slightly lower than calmodulin, was progressively produced. The same species was also detected, under the same conditions, by reacting a Ca-calmodulin and TW31 mixture with the cross-linking reagents (Fig. 5 A, lane e). The 21-kDa band did not appear in the presence of EGTA (data not shown) and when Ca-calmodulin or TW31 was separately treated with EDC (Fig. 5, lanes d and b, respectively). Furthermore, the preformed 21-kDa material failed to co-sediment with added F-actin both in the absence or presence of calcium (Fig. 5 C). These results are consistent with the idea that the 21-kDa material is resulting from a specific conjugation between TW31 and Ca-calmodulin whose reversible or permanent attachment to the peptide interferes with F-actin recognition. This conclusion implies that the sequence Ala-Ileharbors interactive sites not only for F-actin but also for Ca-calmodulin.


Figure 5: EDC cross-linking of the F-actin-TW31 and Ca-calmodulin-TW31 complexes. A, F-actin and TW31 mixed at a 1:8 molar ratio were covalently coupled with EDC-NHS as described under ``Experimental Procedures.'' At the times indicated, the cross-linking reaction was analyzed by 3-18% gradient acrylamide gel electrophoresis in the absence ( lane c) or presence ( lane g) of Ca-calmodulin. Lanes a, b, and d, EDC-NHS-treated F-actin, TW31, and Ca-calmodulin, respectively. Lane e, EDC cross-linking of the Ca-calmodulin-TW31 complex. Lane f, control EDC-treated F-actin-Ca-calmodulin mixture. The 45-kDa and 21-kDa bands represent the 1:1 molar covalent complexes of TW31-actin and TW31-calmodulin, respectively. St = protein markers. B, the electrophoretic pattern of the F-actin-TW31 cross-linking reaction was immunostained with actin antibodies. C, an aliquot of the 30-min cross-linking reaction mixture including Ca-calmodulin and TW31 was supplemented with F-actin (molar ratio actin:TW31 = 1:8) in the absence ( lanes a and b) or presence ( lanes c and d) of 5 m M EGTA and centrifuged for 30 min at 140,000 g. The supernatants ( lanes a and c) and the pellets ( lanes b and d) were examined by gel electrophoresis.



Inactivation of the Acto-S-1 ATPase by the Peptide Analogs of the Sequence Ala-Ileand Its Restoration by the Ca-dependent Proteins

As depicted in Fig. 6 A, TW31 as well as AW19 inhibited significantly the skeletal acto-S-1 ATPase activity in a dose-dependent manner. The presence of tropomyosin in the ATPase assays did not affect their inhibitory potency. In contrast, the three other peptides, TY12, AF18, and QI11, which were unable to co-sediment with F-actin, were inefficient. When the inhibition of ATPase by TW31 was related to the amount of peptide bound to actin, it was found that at least 70% inhibition was reached with about 1 mol of peptide bound/mol of actin (Fig. 6 B). Thus, the actin binding sequence Ala-Ilealso promotes the ATPase inhibition. Moreover, Fig. 7shows that the addition of Ca-calmodulin or Ca-caltropin leads to the reversal of a large extent of the peptide inhibition. In the absence of calcium, no effect was observed at any concentration of either protein (data not shown). As previously reported for the inhibition by native calponin (11) , caltropin was at least twice as efficient as calmodulin in restoring the acto-S-1 ATPase inhibited by AW19. The data clearly indicate that the interaction of the Ca-binding proteins with the stretch Ala-Ileis functionally coupled with the binding of F-actin to adjacent or overlapping sites within the same region.


Figure 6: Inhibition of the acto-S-1 ATPase activity by the synthetic peptides TW31 and AW19. A, ATPase assays were carried out, as specified under ``Experimental Procedures,'' using increasing concentrations of TW31 (), AW19 (), TY12 (), QI11 (), or AF8 (). For TW31, the acto-S-1 ATPase was also measured in the presence of tropomyosin () (actin:tropomyosin molar ratio = 7:1). 100% ATPase = 0.480 µmol of P/min/mg. B, relationship between the degree of ATPase inhibition and the quantity of TW31 bound to F-actin. Parallel determinations were made of the ATPase rate and the amount of peptide bound to F-actin after co-sedimentation and analysis of the resulting pellet by quantitative reverse-phase HPLC on a C-8 column as described under ``Experimental Procedures.''




Figure 7: Reversal of the AW19 inhibition of the acto-S-1 ATPase activity by Ca-binding proteins. ATPase assays were carried out as described under ``Experimental Procedures'' using an 8-fold molar excess of the synthetic peptide AW19 over F-actin in the presence of 1 m M CaCland increasing concentrations of calmodulin () or caltropin (). 100% ATPase = 0.490 µmol of P/min/mg.



Specific Binding of Ca-Calmodulin and Tropomyosin to the Sequence Gln-Ile

To further define the minimal sequence necessary for full binding of the Ca-dependent proteins, we subjected each of the five synthetic peptides to calmodulin-Sepharose affinity chromatography. The experiments were initiated in the presence of 1 m M CaCland under the high ionic strength conditions (0.1 M KCl) we previously employed to assess the interaction of the chymotryptic CH22 fragment with immobilized calmodulin (18) . The overall data are presented in . Only the peptides TW31, AW19, and QI11, which comprise the sequence Gln-Ile(Trp), displayed the capacity to bind to the calmodulin column in a calcium-dependent manner, whereas TY12 and AF8 were not at all retained. The elution of the bound peptides was achieved only in the presence of EGTA and 0.2 M KCl (Fig. 8, A and B), essentially as observed earlier for calponin or the CH22 fragment (18) . These results are in agreement with the above described ability of the Ca-binding proteins to modulate the inhibitory activity of TW31 and AW19. Consequently, the caltropin and calmodulin binding domain on the critical actin interactive sequence Ala-Ileis confined to its COOH-terminal 11-residue segment between Glnand Ile. The spectrofluorometric analysis of the complex between TW31 and Ca-calmodulin did not reveal any change in the tryptophan fluorescence intensity (data not shown).


Figure 8: Binding of the synthetic peptides TW31 and QI11 to calmodulin-Sepharose. The peptides TW31 (0.3 mg) and QI11 (0.1 mg) in 10 m M Tris HCl, 2 m M MgCl, 0.1 m M dithiothreitol, 100 m M KCl, and 1 m M CaCl, pH 7.0, were loaded separately onto a Ca-calmodulin-Sepharose affinity column as specified under ``Experimental Procedures.'' After washing with the same buffer, the column was eluted with 200 m M KCl and 1 m M EGTA added at the point indicated by the arrow. TW31 ( A) and QI11 ( B) were recovered only in the retained fraction eluted by the EGTA-containing buffer.



Because Ca-calmodulin was reported to disrupt the association between calponin and tropomyosin (27, 28) , we also tested in parallel the interaction of the synthesized peptides with immobilized tropomyosin using neutral pH and 10 m M KCl. Interestingly, the data summarized in show that the Ca-calmodulin-binding peptides, TW31, AW19, and QI11 but not TY12 or AF8, were also completely retained by the tropomyosin-affinity column. As illustrated in Fig. 9, A and B, either bound peptide could be eluted by raising the salt concentration of the buffer above 0.1 M. The latter experimental condition was shown earlier to promote the dissociation of native calponin from immobilized tropomyosin (26) . The findings indicate that the sequence Gln-Ilemay also represent a potent tropomyosin binding region. To reinforce this proposal, we compared the relative solution binding of tropomyosin to the different peptides and to native calponin. Because, like caldesmon, calponin interacts near cysteine 190 of tropomyosin (29) , the binding reactions were assayed by measuring the fluorescence changes of pyrene-tropomyosin employed at a constant concentration, at 5 m M MgCland 40 m M KCl (Fig. 9 C). Calponin enhanced the fluorescence by up to 22%, and the change saturated at a calponin:tropomyosin molar ratio of around 3:1; a 35% fluorescence increase was reported for caldesmon under identical experimental conditions (30) . Increasing concentrations of AI19 also enhanced fluorescence by about 70% as much as intact calponin. The maximum fluorescence increase with QI11 was slightly lower accounting for nearly 60% of the calponin effect, suggesting a weaker binding than the longer AI19. In contrast, and as expected, no fluorescence change was noticed on adding TY12. These results indicate that, at the moderate salt concentration used, the changes in fluorescence of pyrene-tropomyosin were not nonspecific but rather due to an interaction between this protein and calponin or the peptide analogs that were found to be retained on immobilized tropomyosin. The influence of the latter peptides on the pyrene environment suggests that, like the parent calponin, they did bind to the region of tropomyosin including Cys.


Figure 9: Interaction of the synthetic peptides TW31 and QI11 with tropomyosin. The peptides TW31 (0.2 mg) and QI11 (0.1 mg) in 10 m M Tris HCl, 10 m M KCl, 1 m M MgCl, 0.1 m M dithiothreitol, and 1 m M NaN, pH 7.0, were loaded separately onto a tropomyosin-Sepharose column as described under ``Experimental Procedures.'' The column was first washed with the same buffer and then eluted with 200 m M KCl added at the point indicated by the arrow. Both peptides TW31 ( A) and QI11 ( B) bound to the immobilized tropomyosin and were recovered only in the fraction eluted by the 200 m M KCl-containing buffer. C, 0.5 µ M tropomyosin labeled with N-(1-pyrenyl)maleimide was mixed with 0-3 µ M calponin () or calponin peptides AI19 (X), QI11 (), or TY 12 () in 5 m M HEPES, 40 m M KCl, 5 m M MgCl, 1 m M dithiothreitol, pH 7.0, at 25 °C, and the fluorescence emission at 377 nm was measured after excitation at 340 nm. Data are expressed as percent increase, as compared with pyrene-tropomyosin alone.




DISCUSSION

In this study, we have used synthetic peptides encompassing the calponin sequence Thr-Ileto demonstrate the direct interaction of F-actin with the 19-residue polypeptide chain between Alaand Ilethat we have earlier assumed to potentially represent the actin binding region of the protein (18) . Two peptide analogs comprising this critical sequence (TW31 and AW19) bound reversibly as well as covalently to F-actin and inhibited the acto-S-1 ATPase activity. In this respect, they accurately mimicked the inhibitory patterns of calponin, CH22, and the active subfragments produced in the limited chymotryptic digest of either of the two latter proteins. The similarity between the synthetic peptide pair and their parent proteins is further underlined by the additional modulation of the inhibitory function of the peptides consequent on their binding to the calcium-dependent proteins, caltropin and calmodulin. We could unambiguously identify the COOH-terminal 11-residue sequence Gln-Ileas an essential stretch for the recognition of these regulatory proteins and the removal of the ATPase inhibition. Most probably, the same region is also serving for the binding of the S100 protein (10, 11) . In the diagram presented in Fig. 10, we designated this 153-163 region as the high affinity caltropin/calmodulin binding site 2 in order to differentiate it from the lower affinity binding site 1 previously localized in the NH-terminal segment of residues 1-52 (19) . The 153-163 peptide would harbor the high affinity site tentatively localized on the segment 52-228 (19) . The juxtaposition and, possibly, the overlapping between CaT/CaM site 2 and the actin binding site within the overall sequence Ala-Ilerationalize the ability of the former site to mediate the restoration of the actomyosin ATPase upon binding to the Ca-dependent proteins. In this regard, calponin resembles caldesmon since both proteins include two binding sites for the Ca-dependent proteins with the higher affinity site being located in the primary structure adjacent to and functionally coupled with an actin binding and inhibitory sequence (20, 31) .


Figure 10: A, diagram illustrating the localization of essential structural determinants which potentially make up the regulatory domain of smooth muscle calponin. These include the identified 145-163 actin-binding and inhibitory segment, the 153-163 stretch representing the tight binding site of the Ca-dependent proteins (caltropin (CaT) or calmodulin (CaM)), and one of the recognition sites of tropomyosin (TM) together with the adjacent Ser/Thr, the phosphorylation of which modulates the calponin-actin interactions (4, 12, 13). The NH-terminal 1-52 sequence houses the low affinity binding site for the Ca-dependent proteins (19) and possibly another tropomyosin interactive region. B, comparison of the amino acid sequences of gizzard ( a) and acidic ( b) calponins in the regulatory 145-163 segment. The sequences are those given for chicken gizzard (3) and rat aorta (37) calponins, respectively.



The functional contribution of the 145-163 sequence is in agreement with our earlier findings describing the cross-linking of actin to the calponin CNBr fragment of residues 52-168 (18) . The inability of AF8 and QI11, which together cover the 145-163 region, to associate separately with actin can be explained by the fact that either a specific actin binding conformation is conferred only by the entire 19-residue-long sequence or that multiple, evenly distributed functional determinants cooperatively interact with actin. The latter possibility would make understandable the moderate loss of ATPase inhibition reported for a calponin recombinant mutated at Lys(32) . On the other hand, recent functional studies on mutant calponin fragments, modified at residues 145-147, revealed the critical importance of these three amino acids for ATPase inhibition.() The latter investigations further substantiate the functional role of the 145-163 sequence in the calponin-actin interactions. Furthermore, the double replacement of Glnand Glnby arginine had no noticeable consequences as we assessed by analyzing a synthetic peptide analog containing these substitutions. In contrast, we observed that the mild treatment of CH22 with the arginine-directed protease, clostripain, leads to the production of the NH-terminal 13-kDa peptide with a severe loss of ATPase inhibition by the digest. This suggests that the structural integrity of the arginyl peptide bonds within the inhibitory 145-163 region is essential for the maintenance of its function. Consequently, the conservation of ATPase inhibition we found with the chymotryptic digest of CH22 should imply the complete lack of proteolytic cleavage of the latter active sequence at the potentially susceptible peptide bond Phe-Gln. This conclusion is consistent with the loss of the inhibitory function we observed when using AF8 and QI11 separately. The higher concentrations of the synthetic 145-163 peptide needed for ATPase inhibition (Fig. 6), as compared to the chymotryptic 145-182 subfragment (Fig. 1), does not reflect a greater inhibitory potency of the latter material since both peptides inhibited the ATPase to the same extent. This quantitative difference most likely results from a difference in the affinity of the two sequences for actin. This proposal is supported by the following two observations. First, a Kof approximately 168 µ M was determined for the complex of actin and the synthetic peptide, indicating an affinity about 100-fold weaker than that of calponin. Second, the investigations on the calponin mutantsshowed that the binding to actin is tighter when the sequence extends through position 182. The recombinant fragment of residues 131-228 was found to display ATPase inhibition with affinity for actin similar to intact calponin. Our conclusions are in agreement with the briefly reported functional properties of a synthetic calponin peptide spanning residues 144-175 which inhibits the acto-S-1 ATPase with a much weaker affinity for actin than calponin (34) .

Our investigations revealed also another striking function associated mainly with the 153-163 segment and consisting in its complexation with tropomyosin. Calponin was earlier shown to bind to the tropomyosin sequence of residues 142-227 (29) , but the tropomyosin-binding site(s) on calponin has not yet been precisely delineated. The calponin 153-163 stretch is certainly not the only tropomyosin binding site of calponin since interaction between tropomyosin and the calponin segment of residues 7-144 was also demonstrated (26) . To account for the occurrence of at least two classes of tropomyosin binding regions on calponin, we have referred, in Fig. 10 A, to the NH-terminally located site as TM binding site 1 and to the 153-163 sequence as TM binding site 2. These two sites do not exclude the interaction of tropomyosin on other additional or complementary stretches. Their juxtaposition to CaT/CaM site 1 and CaT/CaM site 2, respectively, would make tropomyosin and the Ca-dependent proteins compete for binding to calponin. This proposal is consistent with the reported property of Ca-calmodulin or S100 protein to dissociate the calponin-tropomyosin complex (27, 28, 35) . On the other hand, the binding of F-actin to the crucial sequence Ala-Ilecould actually block the interaction of tropomyosin with its binding site 2, thus explaining the absence of tropomyosin effect on the inhibitory capacity of either calponin (36) or the synthetic peptides. So far, it remains to be elucidated whether calponin could firmly attach to tropomyosin in the presence of F-actin.

The amino acid sequence of the multifunctional 145-163 region is highly conserved in all the known smooth muscle calponin isoforms, suggesting that it is important for the biological activity of the calponin molecule. Recently, a novel calponin variant was identified in rat aorta and non-muscle cells such as the central brain, and its covalent structure was established (37) . It differs from the other calponins by the inclusion of extensive changes in the NH-terminal third of the sequence and the addition of a 57-residue acidic domain at the COOH-terminal end. Significantly, this protein was found unable to interact with immobilized calmodulin, a behavior suggesting it has lost the pair of binding sites for the Ca-dependent proteins. While the alterations of the sequence 1-52 may account for the suppression of CaT/CaM binding site 1, Fig. 10 B clearly shows that 6 nonconservative amino acid substitutions are also located on the 153-163 stretch constituting CaT/CaM binding site 2 and are likely responsible for the abolition of its interaction with calmodulin. It is noteworthy that the latter structural changes did not affect the hydrophobic residues of the sequence but rather they caused a neutralization of its net positive charge. Thus, the binding of this sequence to calmodulin and to the other Ca-dependent proteins is not promoted only by hydrophobic interactions as earlier proposed (11, 12) , but it certainly also requires ionic interactions. This proposal is supported by the EDC-catalyzed cross-linking between calmodulin and the 145-163 sequence, we described in this work, and between S100 protein and the CH22 fragment recently reported (35) . Although the NH-terminal segment Ala-Pheis still well conserved, the eventual impact of the COOH-terminal amino acid replacements on the actin recognition by the overall sequence Ala-Valis unknown. However, the recently identified co-localization of the acidic calponin and actin in neuronal cells (39) () and in various other non-muscle tissues (39) would argue for a normal in vivo complexation between the two proteins.

Finally, the 145-163 region we have characterized exhibits reciprocal structural and functional relationships with the adjacent residues of Serand/or Thrwhich are thought to represent the major sites of the in vitro phosphorylation of calponin (12, 13) . The phosphorylation event not only inhibits the binding of calponin to actin (4) but also it significantly lowers its affinity for calmodulin (40) and for tropomyosin (13) without suppressing the interaction of calponin with the two latter proteins (13) . These effects obviously reflect the phosphorylation-mediated disruption of the functioning of the actin site as well as CaT/CaM binding site 2 and TM binding site 2 within the vicinal 145-163 sequence. In contrast, the phosphorylation had no apparent influence on the activity of CaT/CaM and TM binding sites 1 which remain operant in phosphorylated calponin, thus explaining the reported binding of the latter protein to immobilized calmodulin or tropomyosin (4) . Conversely, the binding of calponin to either F-actin, calmodulin, or tropomyosin was shown to inhibit the phosphorylation reaction (13, 33, 41) . The latter effect, which embodies the three interactive functions of the 145-163 sequence, is in agreement with our previous findings illustrating the ability of F-actin to protect the peptide bound at Tyr-Glyagainst proteolysis (18) . Owing to its various interaction properties which are sensitive to calponin phosphorylation at least in vitro, we anticipate that the 145-163 region may represent an important regulatory domain of the protein. It lies just in between the 13-kDa NH- and 13-kDa COOH-terminal fragments that we have earlier proposed to constitute two different structural domains of calponin (18) . Such an interdomain location of the actin binding site was also observed in some other actin-associated proteins like villin (38) . It could confer to the 145-163 sequence specific conformational features relevant to the expression of its multiple functions.

  
Table: Interaction of the synthetic peptides with immobilized calmodulin or tropomyosin

Each peptide was submitted to calmodulin-Sepharose or tropomyosin-Sepharose affinity chromatography as described under ``Experimental Procedures''; the sign (+) means that the peptide exhibits reversible binding to the column whereas the sign (-) indicates that the peptide elutes in the flow-through fraction.



FOOTNOTES

*
This research was supported by grants from CNRS, INSERM, and the Association Franaise contre les Myopathies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a scholarship from the Moroccan Government and the Association Franaise contre les Myopathies.

To whom correspondence and reprint requests should be addressed.

The abbreviations used are: EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide; HPLC, high performance liquid chromatography; CaT, caltropin; CaM, calmodulin; CaP, calponin; TM, tropomyosin.

M. Mezgueldi, P. Strasser, K. Anderson, M. Jaritz, A. Fattoum, and M. Gimona, submitted for publication.

A. Represa, H. Trabelsi-Terzidis, M. Plantier, A. Fattoum, I. Jorquera, F. Dessi, G. Barbin, Y. Ben-Ari, and E. Der Terrossian, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Mario Gimona for communicating prepublication data on the interaction of recombinant calponin fragments with calmodulin and caltropin. We also thank Dr. J. P. Capony for amino acid analyses.


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