pete with whole InI or a In! inhibitory peptide for binding ...... Trewhella, J., Blumenthal, D. K., Rokop, S. E., and Seeger, P. A. (1990). Small-angle scattering ...
REVIEW
In a wide variety of cellular settings, frm organelle transport to muscle contraction, Ca + binding to members of the EF hand family of proteins controls the interaction between actin and different myosins that are responsible for generating movement. In vertebrate skeletal and cardiac muscle the Ca2 -binding protein troponin C (TnC) is one subunit of the ternary troponin complex which, through its association with actin and tropomyosin on the thin ifiament, inhibits the actomyosin interaction at submicromolar Ca2+ concentrations and stimulates the interaction at micromolar Ca2 + concentrations. Because TnC does not interact directly with actin or tropomyosin, the Ca2 +binding signal must be transmitted to the thin filament via the other two troponin subunits: troponin I (TnI), the inhibitory subunit, and troponin T (TnT), the tropomyosinjinding subunit. Thus, the troponin complex is a Ca molecular switch and the structures of and interactions between its components have been of great interest for many years. Although the crystal structure of TnC has been known for almost a decade, the molecular structures of TnI and TnT are not known and therefore convincing models of tle organization of the troponin complex and the Ca +induced changes in its structure have not been forthcoming. Recent advances on a wide variety of fronts including 1) the bacterial expression and characterization of mutants of TnC, TnI, and TnT; 2) cross-linking and fluorescence studies; and 3) the determination of the crystal and nuclear magnetic resonance structures of synthetic and recombinant troponin fragments and complexes between EF hand proteins and their target peptides have provided new insights into the nature of the interactions between troponin subunits. This review discusses these recent advances with the aim of critically2 assessing molecular models of the nature of the Ca +ind d structural transition in troponin.-Farah, C. S., Reinach, F. C. The troponin complex and regulation of muscle contraction. FASEBJ. 9, 755-767 (1995)
ABSTRACT
Key Words: troponin. binding e actomyosin
0892-663819510009-0755/$O1
Tn! .
TnC . Ca2 of muscle
#{149}
regulation
.50. © FASEB
EF hand#{149} Ca2
#{149}
contraction
Muscle fibers are characterized by a highly organized network of parallel thick and thin filaments, polymers of the proteins myosin and actin, respectively (Fig. IA). The actomyosin interaction has been studied extensively in vitro and is controlled in vivo primarily via intracellular Ca2 levels. Studies of the mechanisms by which Ca2 regulates muscle contraction have revealed that the sensors of intracellular Ca2 are members of the EF hand family of Ca2binding proteins: calmodulin (CaM)2 in vertebrate smooth muscle and many invertebrate muscles, the myosin essential light chain (ELC) in molluscan muscle, and troponin C (TnC) in vertebrate skeletal and cardiac muscle and some invertebrate muscles. This, however, is where the recognized similarity among muscular regulatory systems ends, for once Ca2+ is bound, these proteins transmit their signals via clearly distinct mechanisms: Ca2 binding to CaM activates a myosin light chain kinase (MLCK), which phosphorylates the smooth or invertebrate muscle myosin regulatory light chain (RLC), which in turn activates the actomyosin interaction. In molluscan muscle direct Ca2+ binding to the ELC activates muscle contraction. Unlike the above myosin-linked regulatory systems, the contraction of vertebrate skeletal and cardiac muscles, as well as some invertebrate muscle types, is regulated by Ca2 binding to TnC, a subunit of the troponin complex associated with the actinbased thin filament. The troponin-based regulatory system is the best characterized due to the long history of research into the physiology and biochemistry of skeletal muscle contraction (1-5). Structure
of the thin
ifiament
The three principal regulatory components of the skeletal muscle thin filament are actin, tropomyosin, and the troponin complex (Fig. 1B). Actin polymerizes into parallel pseudodouble helical filaments with a pitch of approximately 13 monomers per turn. Tropomyosin molecules,
1To whom correspondence and reprint requests should be addressed, at: Departamento de Bioqulmica, Instituto de Quimica, Universidade de S#{227}o Paulo, Av. Prof. Lineu Prestes 748,01498-970, S#{227}o Paulo, Brazil. 2Abbreviations: CaM, calmodulin; ELC, myosin essential light chain; RLC, myosin regulatory light chain; TnC, troponin C; ml troponin 1; TnT, troponin 1; MLCK, myosin light chain kinase; NMR, nuclear magnetic resonance; Ip, troponin I inhibitory peptide (residues 104-115); Pi, inorganic phosphate.
755
REVIEW head-to-tail overlap region. The troponin complex also interacts directly with actin. Each strand of the thin filament can therefore be thought of as a serial repeat of regulatory units; each unit containing seven actin monomers, one tropomyosin coiled coil, and one troponin complex (1, 2). Troponin has three subunits: troponin I (TnI), which is involved in the inhibition of the actomyosin Mg-ATPase, TnC, which binds Ca2 and removes TnI inhibition, and troponin T (TnT), which binds to tropomyosin (1-4). This complex can be dissociated by denaturants and reassembled to yield a functional complex (6). Functional recombinant troponin subunits and tropomyosins have been expressed in bacteria and it has been demonstrated that the co-expression of all three troponin subunits in the same bacterial cell yields a functional ternary complex that can be purified intact under nondenaturing conditions (7). In this review we discuss recent advances in dissecting the overall organization of the troponin complex, with an emphasis on recent studies using recombinant thin filament proteins, and their implications for our understanding of the mechanism by which this complex regulates muscular contraction.
TROPONIN-TROPOMYOSIN
Figure 1. A) Organization of the sarcomere demonstrating the interdigitation of myosin-based thick filaments and actin-based thin filaments. During muscle contraction these filaments use the energy of ATP hydrolysis to slide pest one another and/or to generate tension along the fiber axis. At the molecular level contractile force is generated when the globular myosin heads or “cross-bridges,” which protrude from the thick filaments, attach to and detach from the actin filaments in a cyclical manner, It is believed that each force-generating cycle is coupled to the hydrolysis of one ATP molecule by the myosin head. This interaction is controlled by levels of intercellular Ca2t B) Model of the troponin-tropomyosin-actin interaction in the absence of Ca2+ where the Ca2+/Mg2f sites in the COOH-terminal domain of TnC are occupied with Mg2, whereas the Ca2-specific sites in the NH2-teiminal domain are empty. In this case the NH2-tenninal region of TnI is bound to the COOH-terminal domain of TnC as well as to the globular COOH-terminal region of TnT, whereas the inhibitory and COOH-terminal regions of ml make contacts with actin and tropomyosin, maintaining the thin filament in a conformation that inhibits the actomyosin interaction. C) Ca2 binding to the Ca2tspecific NH2-terminal sites of TnC increases this domain’s affinity for the inhibitory and COOH-terminal regions of TnI resulting in their release from actin and tropomyosin. This may then allow tropomyosin to adopt a new position on the actin filament thereby liberating the actomyosin interaction that results in contraction. N, NH2-terminal domains of TnC and Tn!; C, COOH-terminal domains of TnC and TnI; I, inhibitory region of TnI.
coiled-coil dimers of 284-residue a-helices, associate with each strand of the actin helix in such a manner that each tropomyosin coiled coil makes contact with seven actin monomers, as well as with neighboring tropomyosins through head-to-tail contacts. Associated with each tropomyosin is a troponin complex that makes contacts along the COOH-terminal third of the coiled coil including the
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Ca2
SWITCH
Muscle fibers lacking troponin and tropomyosin (or mixtures of purified myosin and actin) have relatively high Mg-ATPase activities that are insensitive to Ca2+ concentration. The addition of tropomyosin, which binds to actin cooperatively, either inhibits or enhances this activity, depending on the myosin:actin ratio (8). The addition of purified troponin complex to these systems inhibits the ATPase at submicromolar Ca2+ concentrations and activates the activity at micromolar Ca2+ concentrations (6). It is important to note that troponin’s mechanism of control involves the inhibition of an otherwise strong and favorable interaction between actin and myosin, a characteristic common to systems where a quick, concerted, and highly sensitive response to a stimulus is necessary.
INHIBITION
BY TnI
TnI, a basic globular protein containing approximately 180 amino acids (Mr 21,000), binds to actin, tropomyosin, TnT, and TnC (1, 2). Purified TnI inhibits the actomyosin ATPase on its own in a Ca2+independent manner (6). Actin binding and inhibitory activity are both potentiated by tropomyosin (9, 10). Studies of proteolytic fragments of TnI originally identified the central TnI sequences (residues 96-116 in the rabbit protein) responsible for its inhibitory activity (11). This inhibitory region (Fig. 2) was defined with more precision by Talbot and Hodges (12) who showed that a dodecapeptide corresponding to residues 104-115 (Ip) contains the minimum sequence necessary for inhibition and actin binding. These inhibitory peptides possess approximately
The FASEB Journal
FARAH AND
REINACH
REVIEW half of the inhibitory (11-13). COOH-terminal inhibition
activity
region
of whole TnI on a molar basis
of Tn! also participates
in
Recent studies with recombinant fragments of TnI have shown that fragments lacking the first 57 (TnId57) or 102 residues (TnIlos...182) retain the full inhibitory activity of the entire protein (14, 15). A troponin complex reconstituted with TnIlco...182 inhibits the actomyosin ATPase in a Ca2dependent manner similar to that of wild-type TnI, indicating that sequences on the NH2-terminal side of the inhibitory region of Tn! are not absolutely required for inhibition regulation in vitro (15). Studies with COOH-terminal deletion mutants of TnI revealed that in addition to the inhibitory region, sequences on the COOH-terminal side of the inhibitory region (up to residue 156) are necessary for the full inhibition by TnI alone and the entire COOH-terminal region of TnI may be necessary for full inhibition by the ternary troponin complex at close to physiological troponin:actin ratios (15). These observations suggest that, in addition to the inhibitory region of TnI, sequences on the COOH-terminal side of this region (at least up to residue 156) may be involved in TnI’s interactions with actin and tropomyosin (15). Note that Tn! residues 137-144 have some features in common with residues 108-115 of the inhibitory region:3 residues
108-115:
RPPLRRVR
residues
137-144:
RANLKQVK
This suggests that these two noncontiguous sequences both mediate TnI’s inhibitory activity by interacting actin-tropomyosin in a similar fashion.
Ca2
BINDING
may with
TO TnC
TnC (Mr 18,000) is the Ca2-binding subunit of troponin and is necessary for rendering the actomyosin interaction Ca2+ dependent in systems reconstituted from purified muscle proteins in vitro. When InC is selectively removed from skinned muscle fibers by washing with solutions containing EDTA, the resulting TnC-depleted fibers are permanently relaxed at all Ca2+ concentrations. Addition of exogenous TnC restores Ca2+dependent contraction (16). TnC crystal
structure
The crystal structure of TnC (Fig. 3D) revealed a dumbbell-shaped protein with two globular domains connected by a long central helix (17, 18). Each TnC domain is made up of two EF hand Ca2-binding sites. The EF hand motif consists of two approximately perpendicular cx-helices separated by a 12-amino acid loop, which provides the Ca2-binding ligands. The four TnC Ca2-binding sites are numbered I to IV, according to their order in the primary structure, and the eight a -helices flanking each site are TROPONIN
COMPLEX
lettered A-H from the NH2-terminal to the COOH-terminal (Fig. 3D). Sites I and II in the NH2-terminal domain bind Ca2 specifically with a relatively low affinity of approximately 10 M-’. Sites III and IV in the COOH-terminal domain bind Ca2 with a higher affinity (Ka - 10 M-1) and bind Mg2 with a binding constant of -10 M-1, so that they are occupied by Mg2 under physiological conditions in relaxed muscle (2, 19, 20). Muscle contractioi the low-affinity Ca
triggered by Ca -specific sites
2+
bmding
to
Under physiological conditions, the kinetics of Ca2 binding to the high-affinity COOH-terminal domain sites is expected to be limited by Mg/Ca2 exchange, which is too slow to account for the kinetics of Ca2+induced tension development and relaxation in skinned muscle fibers. On the other hand, the kinetics of Ca2 binding to the empty low-affinity NH2-terminal domain sites is fast (diffusion limited), suggesting that Ca2 binding to sites I and II is the initial event in the removal of inhibition of the actomyosin interaction (2 1-23). TnCs whose COOH-terminal Ca2-binding sites have been inactivated by site-directed mutagenesis can restore Ca2+dependent muscle contraction to TnC-depleted skinned muscle fibers. However, these mutant TnCs are easily removed by washing of the fibers (24, 25). This suggests that the occupation of sites III and IV by divalent metal ions plays a structural role in binding TnC to the thin filament (16). TnC mutants in which sites I or II have been disrupted are deficient in restoring Ca2+dependent contraction to InC-depleted fibers (25, 26). Troponin complexes reconstituted with these mutants inhibit the actomyosin ATPase even in the presence of Ca2 (25). TnC can therefore be divided into a regulatory NH2-terminal domain that binds Ca2 specifically and a structural COOH-terminal domain that binds Ca2 or Mg2 (16, 19,
20). In the TnC crystal structure (17, 18) the two COOH-terminal Ca2+/Mg?+ sites are occupied by Ca2+, whereas the two NH2-terminal Ca2+specific sites are empty, corresponding most closely to the structure in relaxed muscle where the two COOH-terminal sites are expected to be occupied by Mg2. No (Mg2)2-TnC structure has been solved. In the crystal structure the Ca2-loaded COOH-terminal domain has an exposed hydrophobic pocket, which has been proposed as a binding site for the other troponin subunits (27-29). It is not known whether this pocket is also exposed in the presence of Mg and it is therefore not clear whether this proposed site is available under all conditions or only in the presence of Ca2+; i.e., whether it fulfills a structural or a regulatory role in the troponin complex. There is strong evidence from a wide variety of spec-
3.
Chicken skeletal Tnl sequences conserved among species and residue all vertebrate TnIs. Sites of homology are indicated.
. . (92). Residues in bold are highly 138 (underlined) is hydrophobic in (:) and conservative substitution (.)
757
I
I_
V
I I
TV
Tn! .
residues 137-144: homology with inhibitory region and with a calmodulir / binding domain of phosphorylase kinas
1L
\ \ ‘.
.‘4... V
ACTIN-TROPOMYOSIN
residues 96-116: homology with a calmodulin-binding
phosphorylase
COOH-termlnal domain of TnT
domain
of
kinase and lQmotif
t.
residues 53-106: heptad
hydrophobic
repeat
/
)
;k5;_
or
residues 1-40: TnC-binding domain
Mg2 Figure 2.
Organization of the troponin complex. TnI and TnC interact with one another in an antiparallel manner occurring between residues 1-40 of the NH2-terminal region of Tn! and the COOH-terminal domain of InC and the inhibitory and COOH-terminal regions of Tn! and the NH2-terminal domain of TnC as well as between COOH-terminal domain of TnC. Tn! and TnC both bind to the globular COOH-terminal domain of TnT (TnT-2). involves residues 40-98 in the NH2-terminal region of Tn!. N, I, and C represent the NH2-terminal, inhibitory, and of Tn!. Sequences refer to those of chicken-fast skeletal Tn! (92). See text for details.
studies with recombinant TnC mutants containing single tryptophan residues in a variety of locations (30-33) that the Ca2-loaded and Mg2-loaded structures of the COOH-terminal domain of InC are different, though the nature and extent of the difference is unknown. troscopic
Model for the Ca2+induced transition NH2-terzninal domain of TnC
in the
Owing to the significant structural and sequence homology between the two InC domains, Herzberg et al. (34) proposed that on Ca2 binding, the NH2-terminal domain of InC adopts a conformation similar to that observed for the Ca2-loaded COOH-terminal domain in the crystal structure. In this model a hydrophobic surface in the NH2-terminal domain, which is buried when sites I and II are unoccupied (Fig. 3D), becomes exposed to solvent on Ca2+ binding to these sites (Fig. 3E). This is because of a relative movement of helices B and C with respect to helices A and D. This hydrophobic surface in the NH2-terminal domain of InC could, in principle, represent a Ca2-dependent binding site for TnI and/or TnT. 758
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with Ca2-independent interactions Ca2-dependent interactions between the inhibitory region of Tn! and the The TnI-TnT interaction most likely COOH-terminal regions, respectively,
This model has been tested by various groups by using site-directed InC mutants that were designed to stabilize or destabilize the apo- or Ca2-loaded NH2-terminal domain structures in the model. Grabarek et al. (35) showed that a genetically engineered disulfide bridge between helices B and D, which would inhibit the model’s Ca2-induced transition, abolished TnC’s regulatory activity and blocked its ability to interact with TnI. Similarly, Fujimori et al. (36) created a potential salt bridge between the same two helices and found that it reduced the affinity of the NH2-terminal sites for Ca2+. These mutants increased the Ca2+ concentration necessary to trigger tension development in skinned muscle fibers in which the native InC was exchanged for the mutant InCs. Also in accord with this model, InC mutants in which the hydrophobic nature of the NH2-terminal domain hydrophobic pocket was decreased showed an increase in Ca2 affinity at sites I and 11(30, 37). The recently determined nuclear magnetic resonance (NMR) structures of a Ca2+saturated recombinant NH2-terminal domain of chicken InC (residues 1-90) at neutral pH (38) and of whole TnC in 15% trifluoroethanol (Slupsky et a!., Protein Sci., in press) confirm the general features of the
The FASEB Journal
FARAH AND
REINACH
REVIEW
Figure 3. Solid ribbon representation of structures of EF hand protein-target peptide complexes. In all cases the NH2-terminal domains (dark orange) of the EF hand proteins are above the COOH-terminal domains (red). In B-F the target peptides (yellow) have their COOH-terminal end coming out of the page toward the reader while in A the NH2-terminal end of the peptide is above the page. A) CaM-MLCK peptide complex (chicken smooth muscle) (64). B) Essential light chain-heavy chain (residues 773-804) complex (scallop myosin) (67). C) Regulatory light chain-heavy chain (residues 805-836) complex (scallop myosin) (67). Note that while the MLCK peptide (A) is essentially straight, the myosin heavy chain makes a gradual bend of 400 (over six residues, Arg795 to Lys800) where it contacts the NH2-terminal lobe of the ELC (B) and makes a sharper bend of 60#{176} (over three residues Trp4 to Trpns) where it contacts the NH2-terminal lobe of the RLC (C) (67). D) Crystal structure of TnC (17) in which the two NH2-terminal sites (1 and II) are empty and the two COOH-terminal sites (III and IV) are occupied by Ca2. Helices A-H and N are also shown. E) (Ca214-TnC model of Herzberg et al. (34) in which the occupation of two NH2-terminal sites with Ca2 causes this domain to adopt a conformation similar to that of the
COOH-terminal domain. F)
Proposed model of the interaction between Ca2-saturated TnC and the TnI inhibitory region in which structure in E was modified (see text) to approximate that of the regulatory light chain in C and the chicken skeletal Tn! inhibitory (residues 90-121) (92) was modeled on the RLC-binding site of the myosin heavy chain. G) The structure in F rotated about approximately 120#{176} to the left. In F and C the region corresponding to the Ip sequence (residues 104-115) is shown in blue. The inhibitory region is localized to residues 109-111, as in the Ip structure of Campbell and Sykes (28).
model
for the Ca2+induced
transition
in this domain
of
TnC (34).
INHIBIIION
BY In!
IS REMOVED
BY InC
Ihe signal of Ca2 binding to InC is transmitted to the thin filament by way of changes in InC’s interactions with the other troponin subunits (1, 2) (see below). Because TnI binds to both actin-tropomyosin and to TnC, Ca2-induced
TROPONIN
COMPLEX
the (Ca2)4-TnC region sequence the vertical axis bend in the In!
changes in the relative affinities of Tn! (or specific regions of TnI) for actin-tropomyosin and TnC have always been a central theme in understanding regulation by troponin (1-6, 10, 11, 14, 15, 39, 40). TnI and TnC bind with a very high affinity in the presence of Ca2 (Ka 1.7-7.0 x 10 M) and this affinity drops one or two orders of magnitude in the presence of Mg (absence of Ca2) (Ka 0.6-1.2 x 108 M) (41, 42 and refs. therein). In light of the very strong binding observed between TnI and TnC, both in the presence and absence of
759
1%1
V
IlVV
Ca2+, they certainly remain associated under both relaxing and contracting conditions encountered in muscle. Therefore, Ca2+regulated association/dissociation of the TnIInC complex as a whole can be ruled out as a plausible kinetically competent regulatory mechanism. It follows that the subset of Ca2+dependent interactions are at the heart of the regulatory mechanism, whereas Ca2 -independent interactions are involved in maintaining the structural integrity of the complex (1, 2, 4). Tn sites Ca +ddt
mvolved
2+.
m Ca -mdependent interactions with Tn!
and
Weeks and Perry (43), Leavis et al. (20), and Grabarek et al. (44) studied the interactions between fragments of InC and whole InI. These studies suggested that InC has at least three regions that may interact with TnI: metal binding sites II and III of TnC interact with InI in a Ca2-dependent manner, whereas the presence of site IV is necessary for interaction with Ini in the absence of Ca2+. This view was supported by Farah et a!. (15) who used nondenaturing polyacrylamide gels to detect complexes formed at micromolar concentrations of recombinant chicken InC mutants and fragments and recombinant wildtype chicken TnI. Ihey showed that 1) a recombinant NH2terminal domain of chicken skeletal InC (N-InC. residues 1-90) binds to InI in a Ca2+dependent manner, whereas a recombinant COOH-terminal domain of InC (C-InC. residues 88-162) binds to In! in the presence of either Ca2 or Mg2 and 2) a TnC mutant with a nonfunctional site IV has its Tn! binding ability severely reduced in the presence of Mg2+ (absence of Ca2+), whereas mutations that eliminated metal binding to any of the other three sites did not significantly impair binding to wild-type TnI in the presence of Mg24. Swenson and Fredricksen (45) measured the binding constants of InI with different-sized dansylated proteolytic fragments of rabbit skeletal TnC (residues 1-97 and residues 98-i59) in the presence of Ca2+ and Mg2+ and failed to detect a significant difference between the affinities of the two domains for TnI under either condition, though both fragments bound TnI more strongly in the presence of Ca2+ than Mg. As the proportion of InC central helix in each TnC domain differed in these two studies, the anomolous results may reflect a role for the central TnC helix in binding to InI (see discussion and refs cited below). Ca2 + -dependent interactions occur between TnC domains and the inhibitory and COOH-terminal regions of Tn!
both
Syska et al. (11) observed that the proteolytic fragment CN4 (residues 96-116 of TnI) that bound actin and inhibited the actomyosin AlPase also bound to TnC. They therefore suggested that at the heart of the regulatory mechanism is a Ca2-dependent movement of the Ini inhibitory region from actin to TnC. Binding of the inhibitory peptides corresponding to TnI residues 104-i 15 or 96-116 to whole TnC in the presence of Ca2 range from 0.63 to 5.3 x 10 M’, whereas those measured in the presence of Mg or absence 760
Vol. 9
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The
of divalent metals range from 0.7 to 9.5 x 10 M1 (45-48). As this region of InI is expected to be bound to actin-tropomyosin in the absence of Ca2, it may be that only the interaction in the presence of Ca2+ is physiologically relevant. Ihe recombinant TnI fragments TnIlos.1m and InIi.iso, which contain both the inhibitory region (residues 103-116) and residues on its COOH-terminal side (residues 117-156), bind to a recombinant NH2-terminal domain of InC (N-TnC) more strongly than fragments lacking either of these regions (15). On the other hand, an NH2-terminal InI fragment (TnI1_98) does not bind N-InC in the presence of either Ca2+ or Mg2+ at micromolar peptide concentrations. TnI103_lm was also observed to bind to a recombinant COOH-terminal InC domain (C-TnC) but with relatively weaker affinity. The above complexes involving TnIjoi82 with whole InC, N-TnC, or C-InC were only observed in the presence of Ca2+ at micromolar protein concentrations under the nonequilibrium conditions of polyacrylamide gels (containing 10% glycerol or 6 M urea) (15). Therefore, as well as being involved in TnI’s inhibitory activity, both the inhibitory and COOH-terminal regions of TnI participate in its Ca2-dependent interaction with InC (Fig. 2). Both the inhibitory and COOH-terminal regions of TnI, as well as sharing some homologous sequences that may be important for InI’s inhibitory activity (discussed above), possess sequences homologous to the two noncontiguous calmodulin-binding sites of phosphorylase kinase (49). This suggests that these two noncontiguous regions of TnI may bind InC in a manner similar to that observed for CaM and phosphorylase kinase (see ref. 50 for a discussion of possible structural implications) (Fig. 2). Ca2 +independent interactions occur between COOH-terminal domain of TnC and the NH2-terminal region of Tn!
the
Syska et al. (11) were the first to show that proteolytic fragments derived from the NH2-terminal region of In! (residues 1-21 [CN5] and 1-47 [CF2]) bound to TnC (Fig. 2). Initial studies as to the Ca2-dependence of this interaction were inconclusive because some reported a requirement of Ca2+ (13, 51-53), whereas others also observed complex formation in the absence of Ca2+ (11). Recently, conclusive evidence for a Ca2+independent component to this interaction has accumulated as the result of studies with recombinant InIs. Tn! fragments lacking up to 102 NH2-terminal residues (TnId57 and TnIioim) have diminished Ca2-independent interactions with wild-type TnC, whereas the Ca2+dependent interactions are left intact (14, 15).
4Due to an NH2-terminal extension, chicken-fast skeletal InC is three residues longer than rabbit-fast skeletal InC. Therefore, the rabbit TnC fragment 1-97 would correspond to residues 3-100 in chicken TnC and the rabbit TnC fragment 98-159 would correspond to residues 101-162 in the chicken protein (68).
FASEB Journal
FARAH AND
REINACH
REVIEW Farah et al. (15) demonstrated that the NH2-terminal region of Ini binds to the COOH-terminal domain of InC by showing that 1) a complex between TnIi_s and InC forms in the presence of Ca2+ or Mg, 2) these complexes are weakened by mutations that knocked out metal binding to sites III and IV of InC but not by mutations that knocked out Ca2-specific binding to sites I and II, and 3) InIl.98 bound to C-InC in the presence of both Ca2 or Mg2 but did not bind to N-InC in the presence of Ca2 or Mg2. The NH2-terminal region of TnI binds InC with a very high affinity, as witnessed by the fact that the complexes between TnII_98 and recombinant InC or InC COOH-terminal domain can be observed in 6 M urea at micromolar concentrations of both proteins. These same complexes are observed in the presence of Mg2+ only in the absence of urea (15), suggesting that the replacement of Mg2 with Ca2 in the COOH-terminal sites strengthens binding and therefore a Ca2+dependent component to this interaction exists. Fluorimertric titration of recombinant InCs or recombinant InC COOH-terminal domains containing a single tryptophan residue with IflII.98 give association constants of 106-107 rw’ in the presence of Mg2+ and greater than 108 M’ in the presence of Ca2+ (J. R. Pearlstone, C. S. Farah, F. C. Reinach, and L. B. Smillie, unpublished results). Ngai and Hodges (13) showed that a synthetic oligopeptide corresponding to residues 1- 40 of TnI (Rp) can compete with whole InI or a In! inhibitory peptide for binding with InC. Because the site of Rp binding is the COOH-terminal domain of InC and because Rp was able to compete with the inhibitory peptide for binding with InC in the presence of either Ca2+/Mg or Mg2+ (in AlPase assays [13]), the physiological significance of the observed interaction between inhibitory peptides and the COOH-terminal domain of InC remains in question (29, 45, 46). It may be that within the context of native In!, the inhibitory region interacts with the COOH-terminal domain of InC in a manner significantly different from that of small inhibitory peptides, and as Ngai et al. (13, 29) have suggested, may be further affected by Ca2-dependent modulation of the interaction of the NH2-terminal region of TnI with InC. Antiparallel
organization
of the TnI-TnC
interaction
The above observations suggest that the InI regions implicated in its actin binding and inhibitory activity (the inhibitory and COOH-terminal regions) also interact with InC’s regulatory NH2-terminal domain in a Ca2+dependent manner. On the other hand, the Tn! NH2-terminal region interacts with the structural COOH-terminal domain of TnC in the presence of either Ca2+ or Mg. We have therefore proposed (15) that the InI-InC complex is arranged in an antiparallel fashion, as depicted in Fig. 2. In this model the interaction of the regulatory domains of the two proteins requires Ca2+, whereas the two structural domains can interact in the presence of either Ca2+ or Mg. This antiparallel model is consistent with the results of cross-linking studies on the TnI-TnC complex in which TnC was modified with the photoactivatable cross-linker benzo-
TROPONN
COMPLEX
phenone-4-maleimide (approximately 10 A in length). These studies showed that 1) the NH2-terminal half of the TnI inhibitory region (residues 103-113) can be crosslinked to rabbit InC at residue 98 in helix E (54) or residue 89 in the central helix D/E linker (55), 2) the COOH-terminal half of the In! inhibitory region (residues 113-121) can cross-link with TnC residue 57 in helix C (56), and 3) residues 132-141 in the TnI COOH-terminal region can cross-link to TnC residue 12 in the loop between helices N and A (55). In addition, the NH2-terminal residue of the In! inhibitory peptide, G1y103, has been cross-linked to InC’s last three residues at the end of helix H (29); this helix protrudes from the COOH-terminal domain up toward the NH2-terminal domain in the crystal structure of InC and other EF hand protein-peptide complexes (Fig. 3). These cross-linking studies also suggest that the inhibitory region binds to InC in an antiparallel manner (55), similar to that observed in the complexes between other EF hand proteins and their target peptides (Fig. 3 and see below). It is interesting that despite the demonstrated strong interaction between the NH2-terminal region of Tn! and the COOH-terminal domain of TnC, no cross-links demonstrating this interaction have been reported. This is most likely due to the fact that to date no cross-linking experiments have been performed with TnCs labeled in the COOH-terminal domain (other than residue 98 of helix E5) nor with InIs labeled in their NH2-terminal region. Ca2 +dependent COOH-terminal and actin
movement of the inhibitory regions of Tn! with respect
and to TnC
The above considerations seem to indicate that the inhibitory and COOH-terminal regions of InI (especially residues 103-156) (15) are involved in both InI’s inhibitory effect on the actomyosin AIPase and its Ca2+dependent interactions with the regulatory NH2-terminal domain of InC. This dual-binding activity has historically been interpreted as suggesting that a portion of In! makes up the actual switch that moves between actin and InC in a Ca2+dependent manner, thereby regulating some conformational feature of the thin filament. Direct evidence for this kind of movement has been recently provided by fluorescence energy transfer experiments. Tao et al. (40, 57) showed that on Ca2 binding to InC, Cys133 in the rabbit TnI COOH-terminal region moves 1.5 nm away from Cys374 of actin and moves 0.7 nm closer to Cys98 of InC. Figure 1 shows how this movement in the context of the antiparallel nature of the InI-InC complex can be incorporated into a model of the Ca2+in duced transition on the thin filament. In the absence of Ca2 (Fig. 1B) the NH2-terminal Ca2-specific sites of InC are empty, whereas the COOH-terminal Ca2/Mg sites are occupied by Mg2. In this state the inhibitory and
51n one study (54) a significant but small in total) were mapped from Cys of rabbit (residues 22-57) and CB3 (residues 58-81).
fraction of cross-links TnC to Tn! fragments
(30% CN1
761
COOH-terminal regions of InI are bound to actin-tropomyosin, maintaining the thin filament in a conformation in which the actomyosin interaction is weak, whereas the NH2-terminal region of InI (residues 1-40) are bound to the COOH-terminal domain of InC. Ca2 binding to the NH2-terminal domain of InC exposes a hydrophobic pocket, which increases this domain’s affinity for the inhibitory and COOH-terminal regions of In!, causing the movement of these regions away from their binding sites on actin-tropomyosin (Fig. 1C). This would then allow a structural change in the thin filament that activates the actomyosin AlPase.
MOLECULAR MODELS OF INHIBITORY REGION BINDING 10 InC Inhibitory TnC
peptide
is probably
bent
when
bound
to
Campbell and Sykes (28) used two-dimensional NMR spectroscopy to determine the solution secondary structure of the 12-residue inhibitory peptide (Ip) when bound to Ca2loaded InC. They found that the bound peptide has two a-helical regions (residues 104-108 and 112-115) separated by a bend involving two central prolines (residues 109 and 110). Campbell and Sykes (28) proposed that the bend would allow the formation of both a hydrophobic core (involving the side chains of residues PhelO6, ProlO9, Leulli, and Valil4) and a positively charged hydrophillic surface involving residues LyslOS, LyslO7, ArglO8, Argll2, Argll3, and ArguS. To which TnC domain region bind?
does
the TnI
inhibitory
There has been much interest in localizing the site on InC to which the InI inhibitory region (or a particular inhibitory peptide) binds. As mentioned above, the inhibitory region of whole InI and inhibitory peptides have been crosslinked to both the NH2-terminal and COOH-terminal domains of InC. Perturbations in both InC domains induced by inhibitory peptides as small as 12 residues in length have been detected by fluorescence and NMR spectroscopy (45-48, 52). This is, at first glance, surprising in light of the fact that in the InC crystal structure the two domains are separated by a long 31-residue central a-helix (Fig. 3D). This apparent paradox may be resolved by suggestions that, in solution, the central helix of InC bends or partly unwinds, thereby allowing approximation of the two domains. Ihis idea was originally supported by fluorescence energy transfer (58) and low-angle, x-ray-scattering (59) experiments on free InC; however, whether InC is extended or more compact in the troponin complex is still an open question. Recent small-angle, X-ray and neutron-scattering experiments on the Ca2+saturated InI-InC complex in 2-3 M urea suggest a more extendend conformation for InC (60). Studies with deletion mutants of the InC central helix between residues 87 and 97 have revealed that dele-
762
Vol. 9
June 1995
tions of between two and seven residues do not seriously impair the regulatory function of InC, though some differences in the inhibition and Ca2 activation by their reconstituted troponin complexes were observed (61-63). Therefore, the particular distance and relative orientations of the two InC domains observed in the crystal structure (Fig. 3D) do not seem to be important features of InC’s regulatory mechanism. Grabarek et al. (44) suggested that residues 89-100 of the rabbit InC central helix constitutes one of two Ca2-dependent sites for interaction with In! because they observed that only InC fragments containing these residues were able to regulate actomyosin AlPase activity when complexed with InI and TnI, presumably by way of binding to the inhibitory region of In! in the presence of Ca2. Taken together with the cross-linking evidence (above), it seems that the central InC helix may make up at least part of the binding site for the In! inhibitory region. Though they did not directly determine the Ip-binding site on InC, Campbell and Sykes (28) suggested that the Ip hydrophobic surface could be making contacts with the exposed hydrophobic pocket in the COOH-terminal domain of InC. Ngai et a!. (29) cross-linked GlylO4 of the inhibitory peptide to residue 156 of InC and modeled the inhibitory peptide binding to the hydrophobic pocket of the COOH-terminal domain of InC. This model, however, does not address possible contacts of the inhibitory peptide with the NH2-terminal domain of InC. Also, it is not immediately clear how the dissociation of the inhibitory domain from actin and its binding to the COOH-terminal domain of InC, presumably dependent on MgCa2+ exchange at sites III and IV, could occur fast enough to account for the kinetics of Ca2-induced tension development under physiological conditions (2 1-23; discussed above). Clues obtained complexes
from
other
EF hand
protein-peptide
In the last few years crystal and solution structures of three other EF hand proteins, CaM (64, 65), and the two myosin light chains (66, 67) in complexes with their respective target peptides have been solved (Fig. 3A-C). In all these complexes the central helices of the EF hand Ca2-binding proteins are bent or disordered, decreasing the distance between the NH2- and COOH-terminal domains and in some cases allowing interdomain contacts. In the case of CaM in complex with a myosin light chain kinase (MLCK) peptide, the bound peptide adopts a helical conformation throughout its length. In these complexes the NH2- and COOH-terminal domains of CaM make contacts with the COOH- and NH2-terminal halves of the MLCK peptide, respectively (Fig. 3A). Though the antiparallel nature of this interaction is as proposed for the InI-TnC complex (15, 55), the fact that the MLCK peptide is straight while the inhibitory region of In! is expected to be bent suggests that the inhibitory domain will bind to InC in a manner rather different from that observed in the CaM-MLCK peptide complex.
The FASEB Journal
FARAH AND
REINACH
REVIEW The crystal structures
of the chicken skeletal muscle domain (66) and the scallop myosin regulatory domain (67) have revealed how the essential and regulatory myosin light chains (ELC and RLC) bind to the neck region of the myosin heavy chain. In both structures the light chains bind to different parts of a long a-helical stretch of the heavy chain that connects the myosin head to the filainentous myosin rod. Interestingly, the two types of light chains bind in different manners. The ELC binds to a relatively straight and helical portion of the heavy chain with a gradual 40#{176} bend at its COOH-terminal end (Fig. 3B). The RLC, however, binds to an a-helical region of the heavy chain that contains a sharp 60#{176} bend (Fig. 3C). The central RLC helix is kinked and unwound in the center thereby allowing approximation of the two domains, though to a lesser degree than that observed in the ELC-heavy chain or in the CaM-MLCK complexes. This lower degree of approximation of the two RLC domains may be due to their different relative orientations when forced to bind a sharply bent a-helix as opposed to a straight one. The bent structure of the heavy chain at the RLC binding site is reminiscent of the bent inhibitory peptide structure when bound to TnC (28). myosin
motor
heavy
chain
residues
the conserved tion as Irp824
805-836
(67).
This
alignment puts In! proline at position 109 at the same posiin the bend of the myosin heavy chain7. This
model of the In! inhibitory region was docked with the InC model in a manner similar to the RLC-myosin heavy chain complex. In the resulting model of the InC-Tn! inhibitory region complex (Fig. 3F and G) the helical In! inhibitory region on the NH2-terminal side of the bend (residues 90-107) fits into the hydrophobic pocket of the COOH-terminal domain of InC and extends up toward the NH2-terminal domain alongside the partially unfolded InC central helix. The bend in the inhibitory region and the residues to its COOH-terminal side (residues 108-121) make contacts exclusively with the NH2-terminal domain of InC. This
bend redirects the COOH-terminal portion of the inhibitory region a-helix so that it fits into the hydrophobic pocket of the NH2-terminal domain of InC without necessitating a very close approximation of the two InC domains. Although this model is consistent with the results of InI-InC crosslinking and domain-binding studies discussed above, we would like to caution that it is conceptual in nature and is presented here chiefly as a means of illustrating how InC and the In! inhibitory region may interact in a manner
similar to that of well-characterized EF hand protein-peptide complexes.
Molecular model of the inhibitory region of Tn! bound to TnC in a manner similar to that observed in the complex between the myosin heavy chain and RLC
Low resolution
interaction
may serve as a basis for the construction between TnC and region of Tn!. Supporting this idea is the
of a molecular model of the interaction
the inhibitory observation that the 14-residue sequence beginning at position 98 in the inhibitory region of In! (KLFDLRGKFKRPPL) has a degree of homology with the !Q motifs (consensus IQXXXRGXXXRXX4, where X is any amino acid and (I) is a bulky hydrophobic residue [67])
found in a variety of myosin light chain and CaM-binding sites. We began building mensional structures
of the TnI-TnC
complex
Recently, Olah and Irewhella
The above observations suggest that the RLC-myosin heavy chain
model
(69) proposed a model structure of the Ca2+saturated InI-InC complex based on the dimensions and relative orientations of the two proteins within the complex as deduced from low-angle, X-ray and neutron-scatterring experiments (60). In this model both proteins adopt highly elongated structures whose centers of mass are almost coincident (within 10 A) and whose long axes are aligned in a parallel (or antiparallel) fashion in the
complex. The InC central helix is fully extended and the NH2- and COOH-terminal domains oriented with respect to each other as in the model of Herzberg et al. (34) (shown in Fig. 3E). In! is even more extended than InC, with two
such a model by comparing the 3-diof the NH2-terminal domains in the
(Ca2)4-InC model (34) (Fig. 3E) and in the regulatory light chain in the crystal structure of the scallop myosin
able, despite the fact that helix C is four amino acids (approximately one turn) shorter in the regulatory light chain ()6. The COOH-terminal domains of both proteins may also
be superimposed.
The helical
linker
connecting
the
two globular domains of InC is three amino acids shorter and nonhelical in RLC, making modeling of the this part of TnC difficult.
InC residues
88-94
of this linker
region were
therefore modeled as an extended loop so as to bring the two globular
domains
in RLC. Finally, 90-121)
TROPONIN
in the same respective
the In! inhibitory
was modeled
COMPLEX
on the conformation
orientations
A, B, and C superimposed, the D helices of the two proteins NH2-terminal domains at slightly different angles (differleading to poor overlap. We therefore manipulated the and V angles of InC residue 79 at the beginning of the D helix to superimpose it with the D helix of RLC. This manipulation resulted in steric clashes between the D helix and N helix of TnC, which were removed by manipulating the and ji angles of InC residues 14 and 15 in the loop between helices N and A. Chicken and turkey InC sequences are 98% identical (68). 7This structural alignment was chosen to best superimpose the bending residues in the structures of the inhibitory peptide when bound to InC (28) and the myosin heavy chain when bound to RLC (67) and is not the same as the sequence alignment between the In! sequence and IQ motifs. This is not necessarily unexpected because the two scallop myosin heavy chain IQ motifs, which bind the ELC and RLC (Figs. 3B and 3C), bend to different degrees at their COOH-terminal ends (67). 6With helices exit the globular ence of 35_400),
regulatory domain (67) (Fig. 3C). This revealed that helices A, B, and C of the two proteins are virtually superimpos-
as
region (residues of the myosin
$
763
REVIEW structurally 40-50
similar
residues)
“toroidal”
domains
connected
by an
(the -7-nm
first central
and
last helix
(residues 50-130). In this model the InI’s mass is distributed equally over the two halves of InC: the InI NH2- and COOH-terminal domains “cap” the outer surfaces of the two InC domains, much like a halo, whereas the central InI helix spirals along the full length of the InC structure, making contacts with the InC central helix and the exposed hydrophobic pockets of both InC domains. The InI inhibitory region (residues 104-115) would make up part of this spiral, though the specific domain of InC with which it makes contacts was not specified. Because these inhibitory residues
lie just
to the COOH-terminal
side
of the central
Tn! residue (residue 90), an antiparallel orientation of the two proteins would imply that the inhibitory region would interact predominantly with the NH2-terminal domain of InC.
SPREADING THE SIGNAL IROPOMYOSIN
VIA TnT AND
The organization of the thin filament (1, 2) (Fig. 1) has important implications for the mechanism of control because Ca2+ binding to one troponin complex presumably controls the structure of seven actin monomers via tropomyosin. Neighboring troponin complexes do not contact each other nor do they contact more than half of the seven actin monomers within each regulatory unit. Even so, Ca2+ binding to thin filaments is cooperative (70) and troponin presumably inhibits all seven actin monomers’ interactions with myosin in the absence of Ca2+. Because tropomyosin forms a continous filament along each strand of the actin double helix, these observations suggest that information may be propagated along (and/or across) the thin filament between regulatory units via tropomyosin and/or actin. TnT links
the troponin
complex
to tropomyosin
TnT (Mr 31,000) is a structurally assymmetric protein. Its globular COOH-terminal domain (InI-2, residues 159-259) mediates its interactions with Tnl and InC (Figs. 1 and 2), as well as binding near residue Cysl9O of tropomyosin. TnT’s NH2-terminal domain (TnT-i, residues 1-158) lies extended along the COOH-terminal third of the tropomyosin coiled coil from Cysl9O to the COOH-terminal of tropomyosin where it overlaps the NH2 terminus of the adjacent tropomyosin molecule (Fig. 1) (1, 2). TnT’s intimate interactions with tropomyosin has led to suggestions that it may be involved in the cooperativity of tropomyosin binding to actin and/or Ca2 binding to the thin filament.
TnT’s
role
in the regulatory
Ca2+dependent control activity by the InI-InC
764
Vol. 9
June 1995
mechanism
of actomyosin-tropomyosin ATPase binary complex has only been oh-
served at TnI:actin ratios significantly greater than the physiological value of 1:7 (7, 14, 15, 71), though the actual amount of In! bound per seven actin monomers has never been quantified under the same conditions in parallel.8 Potter and Gergely (10) found that in the absence of TnT InC can remove InI from its actin-tropomyosin-binding site in a Ca2+independent manner when the InI:actin ratio is -1:7. TnT was necessary to maintain the troponin complex bound to actin-tropomyosin under these conditions. In addition, the maximum AlPase activity observed in a mixture containing actomyosin-tropomyosin-InI-InC +/Ca2 is approximately equal to that of actomyosin-tropomyosin, whereas the addition of TnT both increases inhibition in the absence of Ca2+ and activates actomyosin AlPase activity in the presence of Ca2 to levels above that of actomyosintropomyosin alone (6, 7, 15). Therefore, TnT may be doing more than just increasing the affinity of troponin for the thin filament as it seems to be involved in 1) spreading the inhibitory effect via tropomyosin to the seven actin monomers in the absence of CaZ+, 2) removing the inhibitory effect from all seven actin monomers in the presence of Ca2, and 3) activating the actomyosin AlPase to levels above those of actomyosin-tropomyosin. These observations suggest that the mechanism of inhibition by InI or regulation by the InI-TnC binary complex may be different from that effected by the more physiologically relevant ternary troponin complex. Some groups have reported that the interaction between InC and TnT is stronger in the presence of Ca2+ than in the presence of Mg2+ (72), whereas others have failed to detect a significant difference (41, 42). Similarly, it is still not clear which domain of InC binds to TnT (1, 2, 44). The NH2-terminal half of TnT, which extends along the COOHterminal third of tropomyosin, does so in a manner independent of Ca2 binding to InC (73). Residues 70-158 of InI seem to be directly involved in this interaction, whereas the first 70 residues appear to be expendible (73-75). The COOH-terminal half of InI, which binds to InI and InC as well as tropomyosin, has its binding to tropomyosin diminished by Ca2 binding to InC (76). Chemical protection and cross-linking studies have shown that TnT binds to a region within residues 40-100 in the NH2-terminal half of In! in a Ca2-independent manner (77, 78 and Fig. 2). Furthermore, Farah et al. (15) showed that the absence of this stretch of residues in InI destabilizes InI’s incorporation into a tertiary troponin complex in the presence of Ca2+ in vitro. Ihis suggests that InI’s interaction with In! may be of prime importance in the organization of the entire complex. Cheung et al. (42) showed that the magnitude of the negative free energy of
8Various parameters of the in vitro actomyosin ATPase assays, such as the type of myosin or myosin fragment used, ionic strength, pH, and both the absolute and relative concentrations of each component, would be expected to greatly influence the regulatory properties of this complex
system.
The FASEB Journal
FARAH AND
REINACH
REVIEW formation of the ternary troponin complex is less than that expected from the measured free energies of formation of the three binary complexes (InI-InC, InI-TnI, and InIInC). This means that the subunits interact less tightly in the ternary complex than observed in the binary complexes. These workers suggested that this relative destabilization of binding may be important to facilitate the Ca2-induced structural transitions in the troponin that initiate the disinhibition of the actomyosin interaction. As first noted by Pearistone and Smillie (79), residues 53-106 of TnI and the COOH-terminal domain of TnT (residues 205-255) possess strong heptad hydrophobic repeats (InI residues 155-205 also posses a weaker, though conserved, heptad repeat). Ihis suggests that In! and InI may be interacting by means of a coiled coil. Note that the InI-binding region of In! is located in between two InI sequences (residues 1-40 and 104-115) whose binding to InC are strengthened in the presence of Ca2 (Fig. 2). It is an attractive hypothesis that a Ca2-induced change in the InI-InC interaction may be transmitted to TnT in part by way of changes in a coiled-coil interaction between In! and InI. This in turn may influence TnT’s interactions with tropomyosin. Any such model, however, would have to explain why NH2-terminal deletion mutants of InI, whose InI-binding properties have been diminished, still regulate the actomyosin Mg-AIPase (15, 80). Potter et al. (80) have postulated that InC must therefore be interacting directly with InI and not simply through InI. Tropomyosin Because no stable interaction between troponin and myosin has been demonstrated, troponin must regulate the actomyosin interaction through control of some central conformational feature of the thin filament. One idea that has dominated the field for more than 20 years (81-83) is that troponin controls the position of tropomyosin on the actin filament: in the absence of Ca2+, tropomyosin would be positioned such that one step in the actomyosin-AlPase cycle is inhibited, whereas in the presence of Ca2 a shift in tropomyosin’s position would remove the inhibition (Fig. 1). Whether inhibition occurs at the actin-binding step and/or subsequent to actin binding is still a matter of controversy (5). Studies of tropomyosin’s interactions with actin and troponin by site-directed mutagenesis have been hampered by the fact that the unique chemical difference between native and recombinant tropomyosin, acetylation of the a-amino group in the native protein but not in the bacterially expressed protein, is of prime importance for tropomyosin’s head-to-tail polymerization and its interactions with actin. Unlike native tropomyosin, unacetylated recombinant tropomyosin does not undergo head-to-tail polymerization and binds very weakly to actin (84). Large NH2-terminal fusions restore polymerization and actin binding but impair the Ca2-sensitive regulatory properties of reconstituted thin filaments (85, 86). Controlled proteolytic digestion of tropomyosin’s COOH-terminal residues also impairs polym-
TROPONIN
COMPLEX
erization and actin binding (87, 88). Monteiro et al. (86) recently produced a recombinant tropomyosin with a small dipeptide I’H2-terminal fusion (ASImy) that was found to be virtually indistinguishable from native tropomyosin in terms of polymerization, affinity for actin, and regulatory properties. It is hoped that this recombinant can be used in the future to design tropomyosin mutants to help map its interactions with actin and troponin. Iroponin restores the ability of these nonnative tropomyosins to bind actin (86, 89). Willadsen et al. (90) and Butters et al. (91) have studied this phenomenon in more detail and found that troponin restores the actin-binding capabilities of these tropomyosins by increasing the intrinsic actin-binding affinities and not through changes in the cooperativity of binding. Ihe same results were obtained by using troponins reconstituted with a TnT lacking the first 69 residues (InI7o.259). A TnT lacking its first 158 residues did not restore actin-binding capabilities, suggesting that residues 70-158 of InI play a role in anchoring tropomyosin to the thin filament (90). Perhaps Ca2-induced changes in the globular portion of the troponin complex can be transmitted to residues 70-158 of TnT, thereby modulating its interactions with tropomyosin. Pearlstone and Smillie (76) suggested that the InI-InC-TnI-2 complex detaches from actin-tropomyosin on Ca2+ binding, remaining attached only via the Ca2-independent interactions between TnT- 1 and tropomyosin. No doubt experiments using mutants of recombinant InIs are under way to investigate the means by which TnT participates in troponins’s regulatory mechanism.
CONCLUSIONS
AND FUTURE
PERSPECTIVES
Iroponin and tropomyosin constitute a molecular switch that regulates muscle contraction by controlling some aspect of thin filament structure vital for its interaction with myosin. Whether this is achieved by way of regulating access to a specific myosin-binding site on actin and/or by controlling the kinetics of ADP and Pi release from myosin already bound to actin is still to be determined. Ihis switch
is permanently eration
does
macromolecular
assembled not depend
in the thin filament on the diffusion
components
and its op-
or assembly
of
into the system. The specific
conformational changes in troponin associated with Ca2+ binding at the heart of this process have yet to be fully elucidated but seem to initially involve the exposure of a hydrophobic pocket in the NH2-terminal domain of InC to which the inhibitory and COOH-terminal regions of In! may bind. Ihe subsequent changes in InI-InI and InCInI interactions and their transmission to tropomyosin and actin remain in the realm of speculation at this time. Hopefully, this state of affairs will change in the upcoming years as more structural data are gathered and a second generation of troponin and tropomyosin mutants are designed to rigorously test, discard, and hopefully confirm specific hypotheses as to how this Ca2+ switch regulates muscle contraction.
765
We would like to thank the members of our laboratory for helpful discussions, Carlos H. I. Ramos for first indicating the homology between Tn! residues 108-115 and 137-144, Dr. 0. Herzberg for the coordinates 2+ of the (Ca )4-TnC model, and Dr. C. Cohen for the coordinates of the regulatory domain of scallop myosin.
26. 27.
Function
28.
29.
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23. 24. 25.
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Ohtsuki, I., Maruyama, K., and Ebashi, S. (1986) Regulatory and cytoskeletal proteins of vertebrate skeletal muscle. Adv. Protein Chem. 38, 1-67 Zot, A. S., and Potter, J. D. (1987) Structural aspects of troponin-tmpomyosin regulation of skeletal muscle contraction. Annu. Rev. Biophys. Chem. 16, 535-559 Da Silva, A. C. R., and Reinach, F. C. (1991) Calcium binding induces conformational changes in muscle regulatory proteins. TIBS 16, 53-57 Grabarek, Z., Tao, 1., and Gergely, J. (1992) Molecular mechanism of troponin-C function. J. Muscle Res. Cell. Motil. 13,383-393 Chalovich, J. M. (1992) Actin-mediated regulation of muscle contraction. Pharmacol. Ther. 55,95-148 Creaser, M. L., and Gergely, J. (1971) Reconstitution of troponin activity from three protein components. J. Biol. Chem. 246,4226-4233
30.
Malnic,
33.
B., and Reinach,
F. C. (1994)
Assembly
of functional
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