Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I Hiroyuki Tanaka1, Yuhei Takeya1, Teppei Doi1, Fumiaki Yumoto2,3, Masaru Tanokura3, Iwao Ohtsuki2, Kiyoyoshi Nishita1 and Takao Ojima1 Laboratory of Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Japan Laboratory of Physiology, The Jikei University School of Medicine, Tokyo, Japan Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan Keywords invertebrate; mollusk; regulatory mechanism; troponin; troponin-I Correspondence Takao Ojima, Laboratory of Biochemistry and Biotechnology, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041–8611, Japan Tel ⁄ Fax: +81 138 408800 E-mail: ojima@fish.hokudai.ac.jp Note The nucleotide sequences of cDNAs encoding Akazara scallop 52K-TnI and 19K-TnI are available in DDBJ ⁄ EMBL ⁄ GenBank databases under accession numbers, AB206837 and AB206838, respectively (Received 24 March 2005, revised 13 June 2005, accepted 15 July 2005) doi:10.1111/j.1742-4658.2005.04866.x Vertebrate troponin regulates muscle contraction through alternative binding of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca2+-dependent manner To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI The C-terminal fragment of Akazara scallop TnI (ATnI232)292), which contains the inhibitory region (residues 104–115 of rabbit TnI) and the regulatory TnC-binding site (residues 116–131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase However, it did not interact with TnC, even in the presence of Ca2+ These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin On the other hand, ATnI130)252, which contains the structural TnC-binding site (residues 1–30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC Moreover, the ternary complex consisting of this fragment, troponin-T, and TnC activated the ATPase in a Ca2+-dependent manner almost as effectively as intact Akazara scallop troponin Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnCbinding site to the inhibitory region of TnI Together with the observation that corresponding rabbit TnI-fragment (RTnI1)116) shows similar activating effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin complex but also for Ca2+-dependent activation Troponin is a Ca2+-dependent regulatory protein complex, which constitute thin filaments together with actin and tropomyosin [1] It is composed of three distinct subunits: troponin-C (TnC), which binds Ca2+, troponin-T (TnT), which binds tropomyosin, and troponin-I (TnI), which binds actin and inhibits actin–myosin interaction [2–4] In relaxed muscle, TnI binds to actin and inhibits contraction Upon muscle stimulation, Ca2+ binds to TnC and induces the release of the inhibition by TnI, resulting in muscle contraction To understand the molecular mechanisms of this Ca2+ switching, extensive studies of the structure, function, and Ca2+-dependent conformational changes of troponin subunits have been carried out In vertebrate muscles, TnC has a dumbbell-like shape with the N- and C-terminal globular domains linked by a central helix [5,6] Each domain contains two EF-hand Ca2+-binding motifs [7], thus TnC has four possible Ca2+-binding sites, sites I and II in the N-domain and sites III and IV in the C-domain [8,9] Abbreviations TnC, troponin-C; TnI, troponin-I; TnT, troponin-T; IPTG, isopropyl-1-thio-b-D-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4475 Functional regions of molluskan TnI Sites III and IV also show affinity for Mg2+ and are thought to be always occupied by sarcoplasmic Mg2+, whereas Ca2+ binding to site I and ⁄ or II is believed to trigger muscle contraction [10] TnC interacts with both TnI and TnT The TnC–TnI interaction and changes in the interaction upon Ca2+ binding to TnC have been intensively studied as the central mechanisms of Ca2+ switching It has been revealed that TnI has three major TnC-binding sites [11–14], namely a structural TnC-binding site (residues 1–30 in rabbit fast skeletal TnI), an inhibitory region (residues 104– 115), and a regulatory TnC-binding site (residues 116– 131) In the relaxed state, the inhibitory region binds to actin and inhibits actin–myosin interaction [11,12], while in the contractile state, Ca2+-binding to site I and ⁄ or II of TnC causes the exposure of a hydrophobic patch on the surface of the N-domain [15], resulting in hydrophobic interaction between the N-domain and the regulatory TnC-binding site [16] This interaction induces the dissociation of the inhibitory region, which is adjacent to the regulatory TnC-binding site, from actin, resulting in the release of the inhibition and muscle contraction [17] The structural TnC-binding site interacts with the C-domain of TnC in both the relaxed and contractile states, which plays a role in maintaining the structural integrity of the troponin complex [17,18] These switching mechanisms were recently confirmed by crystallographic studies of vertebrate troponins [19,20], which demonstrated that the Ca2+-saturated N- and C-domains of TnC bind to the regulatory and structural TnC-binding sites, respectively, of TnI, and suggested that the C-terminal region of TnI (including the inhibitory region and the regulatory TnC-binding site) exhibits a positional change from actin-tropomyosin filament to the N-domain of TnC in a Ca2+-dependent manner However, a significant discrepancy exists between the above schemes and the structural and functional features of some invertebrate troponins Molluskan TnC binds only one mole of Ca2+ per mole of protein at site IV in the C-domain because of amino acid substitutions at sites I–III [21,22] Nevertheless, ternary troponin complex combined with molluskan tropomyosin can regulate the Mg-ATPase activity of vertebrate actomyosin in a physiologically significant Ca2+dependent manner [21] Moreover, the troponin regulates the ATPase of molluskan myofibril together with a well known myosin light chain-linked regulatory system, especially under low temperature conditions [23] Therefore, the molecular mechanisms of regulation by molluskan troponin are expected to be somewhat different from those described above A previous study revealed that the C-domain of molluskan TnC is 4476 H Tanaka et al responsible not only for Ca2+-binding but also for the interaction with TnI, although the presence of both the N- and C-domains is essential for full regulatory ability [24,25] In the present study, we compared the functional sites of molluskan and vertebrate TnI by using the recombinant fragments of Akazara scallop Chlamys nipponensis TnI and rabbit fast skeletal TnI The results provide evidence that molluskan troponin functions through a mechanism in which the region spanning from the structural TnC-binding site to the inhibitory region of TnI plays an important role Results Escherichia coli expression of TnI-fragments Figure 1A shows a schematic representation of the recombinant TnI-fragments used in this study ATnI52K, ATnI-19K and RTnI are the recombinant Akazara scallop 52K-TnI, 19K-TnI (isoforms; see Experimental procedures section and [27]), and rabbit fast skeletal TnI, respectively ATnI1)128 is the fragment corresponding to the N-terminal extending region of 52K-TnI ATnI130)252 and RTnI1)116 are the fragments, corresponding to the regions spanning from the structural TnC-binding sites to the inhibitory regions of Akazara scallop and rabbit TnI, respectively ATnI232)292 and RTnI96)181 correspond to the regions spanning from the inhibitory regions to the C-termini of these TnI Figure 1B shows an SDS ⁄ PAGE of these purified recombinant proteins ATnI-52K and ATnI1)128 showed anomalously low mobility due to the high fraction of hydrophilic residues in the N-terminal extending region as described previously [26] The initiator Met at the N-terminus was removed by the bacterial cell for all these proteins except for RTnI96)181 Inhibition of Mg-ATPase of actomyosin by TnI-fragments The inhibition of actomyosin-tropomyosin Mg-ATPase by TnI fragments was compared The inhibitory effects of RTnI, RTnI1)116 and RTnI96)181 differed greatly from one another, although all of these proteins contained the inhibitory region (Fig 2A) RTnI1)116 inhibited only 33% of rabbit-actomyosin–rabbit-tropomyosin Mg-ATPase at a : molar ratio with tropomyosin, compared with 82% for RTnI As has been reported previously [18,28,29], weaker inhibitory effects of RTnI1)116 revealed the importance of residues 117–181 for maximal inhibition In particular, residues FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS H Tanaka et al Functional regions of molluskan TnI A B Fig (A) Schematic representation of recombinant TnI-fragments The numbers preceding and following each box indicate the amino acid positions of Akazara scallop 52K-TnI (Swiss-Prot #Q7M3Y3) and rabbit fast skeletal TnI (Swiss-Prot #P02643) The N-terminal extending region of 52K-TnI and the functional regions that have been previously identified in vertebrate TnI are indicated by bars The inhibitory regions are shaded (B) SDS ⁄ PAGE of recombinant TnI-fragments used in this study Each protein (1.5 lg) was run on a 10% (w/v) acrylamide gel Molecular mass markers are also shown (M) 140–148 had been proven to bind to actin-tropomyosin and thus are referred to as the second actin-tropomyosin-binding site [14] Moreover, in our results, the inhibition by RTnI96)181 was the strongest (94% of the ATPase was inhibited), suggesting that residues 1–95 may decrease the inhibitory effects of residues 96–181 On the other hand, Akazara scallop TnI isoforms and their fragments showed somewhat different properties (Fig 2B) ATnI130)252, which corresponds to RTnI1)116, inhibited about 70% of rabbit-actomyosinscallop-tropomyosin Mg-ATPase at a : molar ratio with tropomyosin Moreover, the inhibition by ATnI232)292, which corresponds to RTnI96)181, was weaker (51%) than that by ATnI-19K (88%) or ATnI130)252 Therefore, the effects of the N- or C-terminal region of TnI on the function of the inhibitory region appeared to differ between rabbit and Akazara scallop TnI Interestingly, ATnI-52K showed weaker inhibition (65%) than ATnI-19K, suggesting that FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS the N-terminal extending region of 52K-TnI could decrease the inhibitory effects, although ATnI1)128, which corresponds to the N-terminal extending region, on its own, exhibited neither activation nor inhibition To determine whether the inhibitory effect correlates with the binding affinity to actin-tropomyosin, we examined each TnI for its ability to cosediment with actin-tropomyosin When TnI-fragments were mixed at : molar ratios with tropomyosin, RTnI, RTnI1)116 and RTnI96)181 cosedimented with molar ratios of approximately 0.23, 0.048, and 0.35, respectively, to actin On the other hand, ATnI-19K, ATnI130)252 and ATnI232)292 cosedimented with molar ratios of 0.49, 0.44, and 0.065, respectively, to actin (the extent of the cosedimentation of ATnI-52K could not be determined because it precipitated even in the absence of actin-tropomyosin in a control experiment due to the low solubility) Therefore, the observed difference in the inhibitory effects of TnI-fragments might be 4477 Functional regions of molluskan TnI H Tanaka et al Interactions of TnI-fragments with TnC Fig Inhibition of actomyosin-tropomyosin Mg-ATPase by rabbit (A) or Akazara scallop (B) TnI-fragments The actomyosin-tropomyosin Mg-ATPase was measured at increasing ratios of TnI or TnI-fragments to tropomyosin as indicated on the abscissa The measurements were performed at 15 °C The results were expressed as a percentage of the ATPase activity obtained in the absence of TnI Each point is an average of three determinations (A) RTnI, d; RTnI1)116, n; RTnI96)181, h (B) ATnI-52K, d; ATnI19K, s; ATnI1)128, e; ATnI130)252, n; ATnI232)292, h attributable to the difference in their binding affinities for actin-tropomyosin In addition, ATnI1)128 did not cosediment and remained in the supernatant (data not shown) This suggested that the N-terminal extending region of 52K-TnI was not involved in binding to actin-tropomyosin, although this region showed sequence homology to the N-terminal tropomyosin binding site of vertebrate TnT [26] 4478 We compared the ability of TnI-fragments to form a complex with TnC by alkaline urea PAGE The experiments were performed under either or m urea conditions in the presence of either mm EDTA or mm CaCl2 RTnI and both rabbit TnI-fragments formed a complex with rabbit TnC in mm CaCl2 but not in mm EDTA under both urea conditions (Fig 3A) These results agreed with those reported by Farah et al for chicken skeletal TnI-fragments [18], and were compatible with the fact that all of these proteins have at least two of three known TnC-binding sites, namely the structural TnC-binding site, the inhibitory region, and the regulatory TnC-binding site On the other hand, ATnI1)128 and ATnI232)292 did not form a complex with Akazara scallop TnC under any of the tested conditions, whereas ATnI-52K, ATnI-19K, and ATnI130)252 did under both urea concentrations in the presence of Ca2+ (Fig 3B) It was interesting that ATnI232)292 did not form a complex, as ATnI232)292 corresponds to RTnI96)181 and should have two TnC-binding sites, the inhibitory region and the regulatory TnC-binding site Therefore, this suggests that TnC-binding affinities of these regions of the Akazara scallop TnI were much weaker than those of rabbit TnI Moreover, under the m urea condition, ATnI-52K, ATnI-19K, and ATnI130)252 showed complex formation even in the absence of Ca2+ (Fig 3B, upper panels), suggesting that in the absence of Ca2+, the Akazara scallop TnI binds to TnC more strongly than rabbit due to the properties of the interaction between residues 130–252 and TnC We also performed affinity chromatography to confirm the interaction of TnI-fragments with immobilized rabbit or Akazara scallop TnC under nondenaturing conditions (Fig 4) ATnI232)292 binding to Akazara scallop TnC was not observed, even in the absence of both urea and KCl and the presence of 0.5 mm CaCl2, whereas ATnI130)252, RTnI1)116, and RTnI96)181 strongly bound to TnCs These results suggested that the inhibitory region and the regulatory TnC-binding site of Akazara scallop TnI essentially cannot interact with TnC Ca2+-dependent alternative binding of C-terminal TnI fragments to actin-tropomyosin and TnC To understand the biological significance of the difference in TnI–TnC interactions, we compared the ability of TnC to neutralize the inhibitory effects of the C-terminal fragments in the presence and absence of Ca2+ As has been reported for similar vertebrate TnI fragments [14,18,29], the inhibitory effect of RTnI96)181 in FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS H Tanaka et al A Functional regions of molluskan TnI B Fig Complex formation between TnI-fragments and TnC detected by alkaline urea PAGE TnI-fragments were combined with TnC as described under ‘Experimental procedures’ The final concentration of the proteins was 13.8 lM Twenty-microliter aliquots of the mixture were electrophoresed on the gel containing either or M urea and either mM EDTA (– Ca; upper panels) or mM CaCl2 (+ Ca; lower panels) (A) Rabbit TnI or TnI-fragments were run on the gels in the absence (lanes a–c) or presence (lanes d–f) of equimolar amounts of rabbit TnC Lanes a and d, RTnI; lanes b and e, RTnI1)116; lanes c and f, RTnI96)181; lane g, rabbit TnC (B) Akazara scallop TnI or TnI-fragments were run in the absence (lanes h–l) or presence (lanes m–q) of equimolar amounts of Akazara scallop TnC Lanes h and m, ATnI-52K; lanes i and n, ATnI-19K; lanes j and o, ATnI1)128; lanes k and p, ATnI130)252; lanes l and q, ATnI232)292; lane r, Akazara scallop TnC Complex formation was detected by the bands of the TnI–TnC complex (arrowheads) and weakening of the free TnC bands Free RTnI, RTnI1)116, RTnI96)181, ATnI-19K, ATnI130)252, and ATnI232)292 did not migrate into the gels, while free ATnI-52K and ATnI1)128 exhibited a band near the origin and at the middle of the gel, respectively The bands corresponding to the free rabbit or Akazara scallop TnC were found in the middle to bottom of the gels (indicated as RTnC or ATnC, respectively) Fig TnC-affinity chromatography of TnIfragments The fragments of rabbit or Akazara scallop TnI were applied onto the affinity columns prepared by immobilizing either rabbit (A) or Akazara scallop (B) TnC on Formyl-Cellulofine The fragments were eluted with a stepwise gradient of KCl concentrations indicated at the top of the figures Each fraction contains 1.0 mL Eluted protein was detected by the method of Bradford [40] and identified by SDS ⁄ PAGE (data not shown) Due to low solubility, RTnI1)116 was applied at a KCl concentration of 0.1 M a : molar ratio with tropomyosin was effectively neutralized by rabbit TnC in the presence of Ca2+, but not in its absence (Fig 5A, upper panel) In addiFEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS tion, the cosedimentation experiment performed under a : : : molar ratio of RTnI96)181–TnC–tropomyosin–actin showed that the amount of RTnI96)181 4479 Functional regions of molluskan TnI A H Tanaka et al B Fig Functional differences between RTnI96)181 (A) and ATnI232)292 (B) Upper panels, effects of TnC on inhibition by the C-terminal TnIfragments TnI-fragments were present at a : molar ratio of TnI-fragments ⁄ tropomyosin The Mg-ATPase activity was measured at increasing ratios of TnCs to the fragments in the presence (d) or absence (s) of Ca2+ The measurements were performed at 15 °C The results were expressed as a percentage of the ATPase activity obtained in the absence of both TnI and TnC Lower panels, change in C-terminal TnI-fragment affinity for actin-tropomyosin tested by cosedimentation experiments The fragments were added to actin-tropomyosin at a molar ratio of : : (fragment ⁄ tropomyosin ⁄ actin) with or without an equimolar amount of TnC in the presence or absence of Ca2+ The pellets (P) and supernatants (S) were redissolved in equivalent volumes of M urea solution and then run on SDS ⁄ PAGE Lanes a and d, in the absence of both TnC and Ca2+; lanes b and e, in the presence of TnC and the absence of Ca2+; lanes c and f, in the presence of both TnC and Ca2+ Ac, actin; Tm, tropomyosin; RTnC, rabbit TnC; ATnC, Akazara scallop TnC The relative staining intensities of the C-terminal TnI-fragments on lanes a–c were expressed as a percentage of that on lane a and were shown on the right cosedimented with actin-tropomyosin was greatly reduced in the presence of Ca2+ but not in its absence The amount that remained with TnC in the supernatant was greater in the presence of Ca2+ than in its absence (Fig 5A, lower panel) Therefore, this suggested that RTnI96)181 bound actin and TnC in the absence and presence, respectively, of Ca2+ These phenomena should directly reflect the mechanism of Ca2+ switching involving the alternative binding of the C-terminal region of TnI to actin or TnC in a Ca2+dependent manner [17,19] On the other hand, the inhibitory effect of ATnI232)292 was not neutralized by adding Akazara scallop TnC, irrespective of Ca2+ 4480 concentrations (Fig 5B, upper panel) Moreover, the amount of ATnI232)292 cosedimented with actin-tropomyosin was unaffected by the presence and absence of TnC and Ca2+ (Fig 5B, lower panel) Therefore, the Ca2+-switching mechanisms involving the alternative binding of the C-terminal region of TnI were not present in Akazara scallop troponin Ca2+-regulatory effects of troponins containing TnI fragments The Ca2+-regulatory effects of troponins composed of TnI-fragments, native TnT, and TnC on actomyosinFEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS H Tanaka et al Functional regions of molluskan TnI Fig Ca2+-regulation of actomyosin-tropomyosin Mg-ATPase by rabbit (A and C) and Akazara scallop (B and D) reconstituted troponins The effects of the troponin containing TnI or TnI fragments on the actomyosintropomyosin Mg-ATPase were measured as a function of pCa ()10g[Ca2+]) The assays were performed at 15 °C (A and B) or 25 °C (C and D) A and C: RTn, d; RTn1)116, n; RTn96)181, h B and D: ATn-52K, d; ATn19K, s; ATn130)252, n; ATn232)292, h The activities in the absence of troponin are indicated by dashed lines tropomyosin Mg-ATPase were compared The assays were performed at different temperatures, 15 °C, which is the normal ambient temperature for Akazara scallops and is suitable for functionalizing the molluskan troponin [23], and 25 °C, at which many assays of Ca2+ regulation by vertebrate troponin have been conducted [14,18,28–30] At 15 °C, all the ternary complexes consisting of rabbit TnI or TnI fragments, rabbit TnT and TnC, regulated the ATPase, although they exhibited quite different Ca2+-dependence curves (Fig 6A) The complex containing RTnI1)116 (represented as RTn1)116) showed no inhibition, even under low Ca2+ concentrations, although it strongly activated the ATPase at Ca2+ concentrations higher than pCa 4.5 RTn96)181 did not activate the ATPase beyond the level observed in the absence of troponin, even at pCa 4.0 On the other hand, the complex consisting of ATnI232)292, Akazara scallop TnT and TnC (ATn232)292) inhibited the ATPase irrespective of Ca2+ concentration, and could not regulate it at all (Fig 6B) This property could be explained by the fact that the inhibitory region and the regulatory TnC-binding site of Akazara scallop TnI bind to actintropomyosin, but not to TnC, irrespective of Ca2+ concentration, as described above Moreover, ATn130)252 regulated the ATPase almost as effectively as intact troponins (ATn-52K or ATn-19K), suggesting FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS that the region spanning from the regulatory TnCbinding site to the C-terminus of Akazara scallop TnI is not important for this regulation, and that Akazara scallop troponin acts through mechanisms in which the region spanning from the structural TnC-binding site to the inhibitory region plays an important role It should also be mentioned that ATn-52K more strongly activated the ATPase than ATn-19K under high Ca2+ concentrations Thus, the N-terminal extending region of ATnI-52K may be involved in the activation of the ATPase in the presence of Ca2+ When we performed similar assays at 25 °C, the regulation by RTn1)116, which was observed at 15 °C, became unremarkable, whereas RTn96)181 more effectively regulated the ATPase than at 15 °C (Fig 6C) These results obtained at 25 °C were essentially the same as those reported by Farah et al [18] for the chicken skeletal troponins containing similar TnI fragments On the other hand, the regulatory ability of Akazara scallop troponins dramatically decreased (Fig 6D), suggesting that Akazara scallop troponin does not function at the temperature appropriate for vertebrate troponins Discussion The vertebrate TnI is known to interact with TnC in an antiparallel manner such that the regulatory and 4481 Functional regions of molluskan TnI structural TnC-binding sites of TnI interact with the N- and C-domains, respectively, of TnC [18,19] The inhibitory region is known to interact with both the N- and C-domains, but preferentially with the C-domain [18,20,31] In the present study, we revealed a striking difference in the TnI–TnC interactions of vertebrate and mollusk We showed that ATnI232)292, which is the Akazara scallop TnI-fragment containing the inhibitory region and the regulatory TnC-binding site, does not bind to Akazara scallop TnC, whereas ATnI130)252, which contains the structural TnC-binding site and the inhibitory region, strongly binds to TnC The antiparallel structural features of vertebrate TnI–TnC complex and previous observations that the N-domain of Akazara scallop TnC did not bind to TnI while the C-domain bound strongly [24], suggest a single interaction between the structural TnC-binding site of TnI and the C-domain of TnC in Akazara scallop TnI–TnC complex Although the further verification under nondenaturing conditions is required, the results of the alkaline urea gel electrophoresis indicate that this interaction is strengthened by Ca2+ and is stronger than the corresponding interaction in rabbit TnI–TnC in the absence of divalent cation Therefore, this interaction potentially participates in both the Ca2+-dependent activation of the contraction and the maintenance of structural integrity of the troponin complex in the relaxed state Troponin-tropomyosin based regulation exhibits two components [32]: inhibition and removal of inhibition in the absence and presence, respectively, of Ca2+, and Ca2+-dependent activation The regulatory mechanism involving the alternative binding of the C-terminal region of TnI to actin or TnC should be responsible for the former However, it cannot account for the latter, namely the phenomenon that, in the presence of Ca2+, troponin activates actomyosintropomyosin Mg-ATPase beyond the level observable in the absence of troponin This activation is prominent, especially for molluskan troponin, which confers Ca2+ sensitivity on the ATPase predominantly through its activation in the presence of Ca2+, rather than by inhibition due to its absence In contrast, the vertebrate troponin regulates the ATPase mainly by inhibition in the absence of Ca2+ (Fig and [21,32]) The difference in Ca2+ sensitization between vertebrates and mollusks should also be closely related to the difference in the inhibitory effects of vertebrate and molluskan tropomyosins [33], which inhibit rabbit actomyosin Mg-ATPase activity to 0.043 and 0.021 lmolỈmin)1Ỉmg myosin)1, respectively, at 15°C (Fig 6A,B) In the present study, we compared the functional roles of the N- and C-terminal regions of 4482 H Tanaka et al molluskan and vertebrate TnI and revealed for the first time that (a) the alternative binding of the TnI C-terminal region is not observed in molluskan troponin, as the C-terminal region of molluskan TnI does not interact with TnC; and (b) molluskan troponin regulates the ATPase by a mechanism in which the TnI N-terminal region (from the structural TnC-binding site to the inhibitory region) participates in the Ca2+-dependent activation In addition, at 15°C, similar activation is observed for the troponin containing the corresponding vertebrate TnI-fragment, suggesting the presence of a common activating mechanism between vertebrates and mollusks In molluskan troponin, the activation is probably induced by strengthening of the interaction between the structural TnCbinding site and the C-domain of TnC accompanying Ca2+ binding to site IV of TnC In vertebrate troponin, the activation may be a result of the interaction between the inhibitory region and TnC accompanying Ca2+ binding to site I or II of TnC However, we cannot rule out the possibility that the substitution of Mg2+ at site III or IV of vertebrate TnC with Ca2+ causes the activation in vitro Several observations have indicated that the N-terminal region of vertebrate TnI is involved in the activating process [14,28,30] In particular, Malnic et al [30] suggested that the activating effects of the N-terminal region of TnT are exerted in the presence of Ca2+ by the TnI N-terminal region (from the structural TnC-binding site to the TnT-binding site) and TnC In summary, we propose a novel view of the general architecture of TnI In vertebrate muscles, the C-terminal region plays a role in the inhibition ⁄ removal of inhibition by alternative binding, while the N-terminal region is responsible for the Ca2+-dependent activation This view replaces the general and conventional view that the N-terminal region of TnI only plays a role in maintaining the structural integrity of the troponin complex In molluskan muscles, the C-terminal region does not function and troponin regulates contraction only through the activation exerted by the N-terminal region of TnI Experimental procedures Muscle proteins Tropomyosin, TnT, and TnC from Akazara scallop striated adductor muscle or rabbit fast skeletal muscle were prepared by the method of Ojima and Nishita [21,34] Rabbit fast skeletal myosin and F-actin were prepared by the method of Perry [35] and Spudich and Watt [36], respectively All measures were taken to minimize pain and FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS H Tanaka et al discomfort of animals The procedures were conducted in accordance with the institutional guidelines by Hokkaido University Construction of plasmids expressing TnI fragments Based on the partial nucleotide sequence (GenBank accession number AB009368), we cloned the cDNA including the entire coding region for Akazara scallop TnI by 5¢-RACE [37] from the striated adductor muscle As a result, two cDNA clones encoding isoforms, namely 52K-TnI and 19K-TnI [27], were obtained The deduced amino acid sequence of 19K-TnI was identical to that of C-terminal 163 residues of 52K-TnI The 52K-TnI-cDNA was subcloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA), and used as a template for PCR to amplify the DNAs encoding various regions of 52K-TnI For the amplification of the DNAs encoding ATnI-52K (recombinant 52K-TnI; residues 1–292), ATnI1)128 (recombinant fragment consisting of residues 1–128 of 52K-TnI), ATnI-19K (recombinant 19K-TnI; residues 130–292), ATnI130)252 (fragment; residues 130–252), and ATnI232)292 (fragment; residues 232–292), combinations of the forward and reverse primers, ATnI1F (5¢-CATATCACCATGGGTTCCCTTG-3¢) and ATnI292R (5¢-CTTGATTTGGATCCTTTAAGGTA TAGC-3¢), ATnI1F and ATnI128R (5¢-GTTCCGGATC CTATCTTCTGGCTTCC-3¢), ATnI130F (5¢-GCCAGAA CCATGGCGGAGGAAC-3¢) and ATnI292R, ATnI130F and ATnI252R (5¢-CAAGTTTGGGATCCTATTTGTTAA CTTTTC-3¢), and ATnI232F (5¢-CGAGATTAATGCC ATGGCACTTAAGG-3¢) and ATnI292R, respectively, were used These forward and reverse primers introduced NcoI and BamHI restriction sites (underlined), respectively, into the PCR products These primers also introduced the initiation or termination codons (bold), except in ATnI292R, which would anneal to the 3¢-noncoding region It should be noted that in ATnI1F and ATnI232F, the Ser1 and Thr232 codons in the template were replaced by Gly1 and Ala232, respectively, in addition to introducing the NcoI site The PCR products were digested with NcoI and BamHI and then ligated into the NcoI-BamHI site of the expression vector, pET-16b (Novagen, Madison, WI, USA) We also cloned the cDNA encoding rabbit fast skeletal TnI from the back muscle of rabbit by RT-PCR using the primer set, RTnI1F (5¢-CAAACCTCACCATGGGAGAT GAAG-3¢) and RTnI181R (5¢-CCCCGGAGCCGGATCC CCAGCCCC-3¢) These primers were designed based on the sequence retrieved from the GenBank database under accession number L04347, and NcoI or BamHI sites (underlined) and the initiation codon (bolded) were introduced into the sequences The cDNA subcloned into pCR2.1TOPO was first subjected to mutagenesis for deactivating the native NcoI site in the coding region by using MutanSuper Express Km kit (Takara-bio, Ohts, Japan) The FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS Functional regions of molluskan TnI mutated DNA was cut out with NcoI and BamHI and ligated into pET-16b for the construction of the plasmid expressing RTnI (recombinant rabbit fast skeletal TnI; residues 1–181) The expression plasmid was also used as a template for PCR to amplify the DNA encoding RTnI1)116 (fragment; residues 1–116 of rabbit fast skeletal TnI) and RTnI96)181 (fragment; residues 96–181), using the primer sets RTnI1F and RTnI116R (5¢-GAGCATGGCGGGAT CCTACATGCGCAC-3¢) and RTnI96F (5¢-GCTGGAGG CCATGGACCAGAAGC-3¢) and RTnI181R, respectively (BamHI ⁄ NcoI sites and termination ⁄ initiation codons are indicated by underlines and bold type face, respectively) In RTnI96F the Asn96 of the template was replaced by Asp96, and an NcoI site was introduced The PCR products were used for the construction of expression plasmids by the method described above Expression and purification of recombinant TnI fragments The expression plasmids were introduced into E coli BL21(DE3) cells (Novagen) and cultivated at 37 °C for h in LB medium, and then TnI fragments were expressed by induction with mm IPTG The cells were harvested by centrifugation (10 000 g, 10 min), and resuspended in STET buffer (8% (w/v) sucrose, 50 mm Tris ⁄ HCl (pH 8.0), 50 mm EDTA, and 5% (v/v) Triton X-100), and then lysed by three freeze-thaw cycles After centrifugation (10 000 g, 10 min), ATnI1)128, ATnI232)292, and RTnI96)181 were found in the supernatant, and purified by CM-Toyopearl 650 m (Tosoh, Tokyo, Japan) column chromatography in the presence of m urea [34] ATnI-52K, ATnI-19K, ATnI130)252, RTnI, and RTnI1)116, which were found in the precipitate, were dissolved in m guanidine hydrochloride, 10 mm Tris ⁄ HCl (pH 7.6), mm EDTA, and mm 2mercaptoethanol, and then subjected to CM-Toyopeal column chromatography as described above ATnI-52K was further purified by DEAE-Toyopearl 650 m (Tosoh) column chromatography under the conditions used for CMToyopeal chromatography RTnI, RTnI1)116, and ATnI19K were also purified by hydroxyapatite (Wako Pure Chemicals, Osaka, Japan) column chromatography performed using m urea, 10 mm KH2PO4 (pH 7.0), mm 2mercaptoethanol, and a linear gradient of 0–500 mm KCl The N-terminal sequences of these recombinant proteins were analyzed on an ABI 492HT protein sequencer (Applied Biosystems, Foster City, CA, USA) Polyacrylamide gel electrophoresis SDS ⁄ PAGE was carried out using the method of Porzio and Pearson [38] on a 10% (w/v) acrylamide and 0.1% bisacrylamide slab gel Alkaline urea PAGE was performed by the method of Head and Perry [39] on a 6% (w/v) acryl- 4483 Functional regions of molluskan TnI amide and 0.48% (w/v) bis-acrylamide slab gel containing either m or m urea and either mm CaCl2 or mm EDTA The samples were prepared as follows: TnI-fragment and TnC were mixed to a : molar ratio in the medium containing 0.125 m KCl, 10 mm Tris ⁄ HCl (pH 7.6), and either mm CaCl2 or mm EDTA, and then diluted with 1.5 volumes of either 10 or m urea, 41.5 mm Tris, 133 mm glycine (pH 8.6), 0.02% (w/v) bromophenol blue, and 8% (v/v) 2-mercaptoetanol The samples were allowed to stand for h on ice before application to the gels The electrophoresis was carried out at room temperature by using 25 mm Tris and 80 mm glycine (pH 8.6) as a running buffer The gels were stained with 0.2% (w/v) Coomassie brilliant blue R250 Fluorescent staining using SYPRO Red (Cambrex, East Rutherford, NJ, USA) was also performed for densitometric analysis on a fluorescent imager, FLA3000G (Fuji Photo Film, Tokyo, Japan) Affinity chromatography Rabbit or Akazara scallop TnC was immobilized on Formyl-Cellulofine (Chisso, Tokyo, Japan) according to the procedure suggested by the manufacturer The TnCCellulofine was packed into a column (0.8 · 4.0 cm) and equilibrated with 10 mm Tris ⁄ HCl (pH 7.6) and 0.5 mm CaCl2 About 50 nmol of TnI-fragment was dialyzed against the same solution and then applied onto the column The fragment was eluted with a stepwise gradient of KCl at a flow rate of 0.16 mLỈmin)1 The fragment that was not eluted under these conditions was removed with m urea, 0.5 m KCl, 10 mm Tris ⁄ HCl (pH 7.6), and mm EGTA The proteins in the effluents were detected by the method of Bradford [40], and identified by SDS ⁄ PAGE RTnI1)116, which was insoluble in 10 mm Tris ⁄ HCl (pH 7.6) and 0.5 mm CaCl2, was applied at a KCl concentration of 0.1 m Actin-tropomyosin centrifugation studies The binding of the TnI-fragment to actin-tropomyosin was analyzed by a cosedimentation assay The assay conditions were as follows: 0.15 mgỈmL)1 (3.6 lm) rabbit F-actin, 0.075 mgỈmL)1 (1.1 lm) rabbit or Akazara scallop tropomyosin, 2.2 lm recombinant TnI-fragment with or without equimolar amount of TnC, 50 mm KCl, 20 mm Tris maleate (pH 6.8), mm MgCl2, and 0.2 mm EGTA (in the absence of Ca2+) or 0.2 mm EGTA plus 0.3 mm CaCl2 (in the presence of Ca2+) The proteins were mixed in the presence of 0.3 m KCl and then diluted to the above conditions The samples (0.5 mL) were incubated at 15 °C for 30 and then centrifuged at 100 000 g for 30 on an Optima TL-100 ultracentrifuge (Beckman Coulter, Fullerton, CA, USA) The pellets and supernatants were redissolved in equivalent volumes (0.1 mL) of m urea, mm Tris ⁄ HCl 4484 H Tanaka et al (pH 8.9), 0.5% (w ⁄ v) SDS, and 5% (v ⁄ v) 2-mercaptoethanol, and then analyzed by SDS ⁄ PAGE The amount of the TnI-fragment bound to actin-tropomyosin was estimated by densitometry, using known amounts of protein run on the same gel, as a standard The amount of nonspecific precipitation of the TnI-fragment was also monitored by simultaneous centrifugation of the sample containing no actin-tropomyosin under the same conditions Reconstitution of troponins Recombinant TnI-fragment and native TnC and TnT were mixed at a : : molar ratio and dialyzed against m urea, 0.5 m KCl, 10 mm Tris ⁄ HCl (pH 7.6), and mm 2-mercaptoethanol The urea and KCl concentrations were reduced stepwise by the following changes of dialysis buffer: (a) buffer B (3 m urea, 0.5 m KCl, 10 mm Tris maleate (pH 6.8), mm MgCl2, 0.2 mm EGTA, 0.3 mm CaCl2, 0.01% NaN3 (w/v), and mm 2-mercaptoethanol); (b) buffer B containing m urea and 0.5 m KCl; (c) buffer B containing 0.5 m KCl; and (d) buffer B containing 0.25 m KCl After dialysis, the complexes were centrifuged and the supernatants were used immediately Measurements of Mg2+-ATPase activity The inhibition of actomyosin-tropomyosin Mg2+-ATPase by the TnI-fragment and the release of the inhibition by TnC were measured in the presence of 0.05 mgỈmL)1 (1.2 lm) rabbit F-actin, 0.1 mgỈmL)1 (0.19 lm) rabbit myosin, 0.025 mgỈmL)1 (0.38 lm) rabbit or Akazara scallop tropomyosin, and various concentrations of TnI-fragment and TnC The assays were performed at 15 °C in a medium containing 50 mm KCl, mm MgCl2, 20 mm Tris maleate (pH 6.8), mm ATP, and 0.2 mm EGTA (in the absence of Ca2+) or 0.2 mm EGTA plus 0.3 mm CaCl2 (in the presence of Ca2+) The Ca2+ regulatory effect of the reconstituted troponin was measured in the presence of 0.03 mgỈmL)1 (0.71 lm) rabbit F-actin, 0.06 mgỈmL)1 (0.11 lm) rabbit myosin, 0.015 mgỈmL)1 (0.23 lm) rabbit or Akazara scallop tropomyosin, and 0.23 lm reconstituted troponin The assays were performed at 15 or 25 °C in a medium containing 50 mm KCl, mm MgCl2, 20 mm Tris maleate (pH 6.8), mm ATP, 0.1 mm CaCl2 and 0–3.84 mm EGTA The concentrations of EGTA required to attain the desired final free Ca2+ concentrations (pCa 7.5–4.0) were calculated by using the stability constant of 8.45 · 105 m)1 for the Ca2+–EGTA complex [41] The reaction was initiated by adding 0.5 mL of 10 mm ATP to 4.5 mL of the solution containing all the components except for ATP After 2, 4, 6, and incubation, mL aliquots were withdrawn from the reaction mixture and added to mL of acidic malachite green solution to determine the liberated inorganic phosphate concentrations by the method of Chan et al [42] FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS H Tanaka et al Functional regions of molluskan TnI Acknowledgements This study was supported by Special Coordination Funds from the Ministry of Education, Culture, Sports, Science and 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myofibrillar proteins with sodium dodecyl sulphate– polyacrylamide gel electrophoresis Biochim Biophys Acta 490, 27–34 39 Head JF & Perry SV (1974) The interaction of the calcium-binding protein (troponin C) with bivalent cations and the inhibitory protein (troponin I) Biochem J 137, 145–154 40 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 41 Harafuji H & Ogawa Y (1980) Re-examination of the apparent binding constant of ethylene glycol bis (beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid with calcium around neutral pH J Biochem (Tokyo) 87, 1305–1312 42 Chan KM, Delfert D & Junger KD (1986) A direct colorimetric assay for Ca2+-stimulated ATPase activity Anal Biochem 157, 375–380 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS ... 6A,B) In the present study, we compared the functional roles of the N- and C-terminal regions of 4482 H Tanaka et al molluskan and vertebrate TnI and revealed for the first time that (a) the alternative... of the troponin complex In molluskan muscles, the C-terminal region does not function and troponin regulates contraction only through the activation exerted by the N-terminal region of TnI Experimental... amount of nonspecific precipitation of the TnI-fragment was also monitored by simultaneous centrifugation of the sample containing no actin-tropomyosin under the same conditions Reconstitution of