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Call For Papers: Comparative Genomics

Functional and evolutionary relationships of troponin C

¹ Department of Integrative Biology, University of Guelph, Guelph
² The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Ontario
³ Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby
⁴ Cardiovascular Sciences, Child & Family Research Institute, Vancouver, British Columbia, Canada

	ABSTRACT

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
Striated muscle contraction is initiated when, following membrane depolarization, Ca²⁺ binds to the low-affinity Ca²⁺ binding sites of troponin C (TnC). The Ca²⁺ activation of this protein results in a rearrangement of the components (troponin I, troponin T, and tropomyosin) of the thin filament, resulting in increased interaction between actin and myosin and the formation of cross bridges. The functional properties of this protein are therefore critical in determining the active properties of striated muscle. To date there are 61 known TnCs that have been cloned from 41 vertebrate and invertebrate species. In vertebrate species there are also distinct fast skeletal muscle and cardiac TnC proteins. While there is relatively high conservation of the amino acid sequence of TnC homologs between species and tissue types, there is wide variation in the functional properties of these proteins. To date there has been extensive study of the structure and function of this protein and how differences in these translate into the functional properties of muscles. The purpose of this work is to integrate these studies of TnC with phylogenetic analysis to investigate how changes in the sequence and function of this protein, integrate with the evolution of striated muscle.

phylogenetic analysis; protein evolution; temperature; muscle

	INTRODUCTION

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
TROPONIN C (TnC), present in all striated muscle, is the Ca²⁺-activated trigger that initiates myocyte contraction. The binding of Ca²⁺ to TnC initiates a cascade of conformational changes through the component proteins of the thin filament, leading to the formation of cross bridges (CBs) between actin and myosin and the generation of force by the myocyte. Therefore, the functional properties of TnC, including its ability to be activated by Ca²⁺, have significant regulatory influence on the contractile reaction of the myocyte. Myocyte contractility is also influenced by the strength of interaction between actin and myosin, the rate of CB cycling, and the rate of ATP hydrolysis by myosin ATPase (24). There are two muscle-specific TnC proteins found in vertebrate striated muscle. The first, skeletal TnC (sTnC), is expressed in fast skeletal muscle and the second, cardiac TnC (cTnC), is expressed in cardiac and slow skeletal muscle. A critical difference between these two paralogs is that sTnC is activated by Ca²⁺ binding to two low-affinity sites on the NH₂ terminus of the protein (sites I and II), while cTnC is activated by Ca²⁺ binding to a single low-affinity site (site II). Site I is nonfunctional in cTnC due to sequence manipulations that have disrupted its ability to coordinate Ca²⁺ ion binding.

cTnC and sTnC have been cloned from a variety of vertebrate species across a range of phylogenic groups. The most ancient of these is the arctic lamprey, Lampetra japonica, a member of the earliest diverged vertebrate taxon. L. japonica sTnC and cTnC are 70 and 83% identical to human sTnC and cTnC, respectively. The presence of distinct cTnC and sTnC paralogs in L. japonica suggests that these proteins have played a key role in defining, as well as differentiating, the functional characteristics of skeletal and cardiac muscle since the beginning of vertebrate evolution. To date cTnC has been cloned from seven mammalian species, two bird species, one frog species, and 10 species of teleost fish, as well as two lamprey species. There is also one cTnC mutant that has been sequenced from the genomic DNA of a human patient with the pathology hypertrophic cardiomyopathy (HCM). Skeletal TnC orthologs have been cloned from an even larger range of animals including mammals, birds, fish, and lizards. TnC homologs have also been cloned from the skeletal muscle of invertebrates, a tunicate, an ascidian, and a variety of insects and mollusks.

The purpose of the following work is to consider the differences in the sequence, structure, and function of TnC in light of the evolutionary and functional constraints under which the different proteins have evolved. To date there have been a number of studies that have characterized the structural and functional differences between cTnC and sTnC as well as between a number of cTnC paralogs and mutants. This work includes the use of X-ray crystallography and three-dimensional NMR solution studies to solve protein structure and the use of fluorescent probes and two-dimensional NMR to characterize functional properties in solution. In addition, the replacement of native homologs, in both skeletal and cardiac tissues, with recombinant homologs has been used to characterize functional differences in vivo. To examine how the genes of TnC have evolved and to integrate gene evolution with the protein function data, we have completed phylogenetic analysis on the amino acid sequences of all known TnC homologs. Through such integration the following questions will be addressed: 1) How do differences in TnC function relate to differences in muscle function? 2) What is the relationship between TnC sequences and vertebrate evolution? 3) Are there specific residues that are only common to orthologs with specific functional characteristics?

	COMPARISON OF THE STRUCTURE AND FUNCTION OF sTnC AND cTnC

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
cTnC and sTnC are small (161 and 162 amino acids, respectively) dumbbell-shaped proteins composed of two Ca²⁺ binding domains separated by an -helical linker (Fig. 1). Each Ca²⁺ binding domain contains two EF hands, common to other Ca²⁺ binding molecules, composed of a helix-loop-helix structural motif. The low-affinity NH₂-terminal domain contains Ca²⁺ binding sites I and II, while the high-affinity COOH-terminal domain contains Ca²⁺ binding sites III and IV. It has been suggested by Collins (8) that TnC, as well as calmodulin, myosin essential light chains, and myosin regulatory light chains evolved from a common ancestor containing four similar Ca²⁺ binding sites (EF hands) that arose by gene duplication and reduplication. While the functional properties of the Ca²⁺ binding sites of these different proteins have been altered through sequence manipulation, the tertiary structure of the proteins have been largely retained (8). Usually, each EF hand is able to bind one Ca²⁺ ion; however, in cTnC the first EF hand (site I) is nonfunctional due to sequence substitutions.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Ribbon diagram of chicken cardiac troponin C (cTnC) based on NMR solution structure by Sia et al. (51). The relative positions of residues 2, 28, 29, and 30 are indicated on the figure. Ca2+ binding sites and helices are labeled.

When a cardiac or fast skeletal myocyte is relaxed, sites III and IV of TnC are bound by divalent metals (either Ca²⁺ or Mg²⁺). The near NH₂ terminus of TnI is bound to the COOH terminus of both TnC and TnT (31, 33). A section of the TnI molecule known as the inhibitory peptide is bound to two adjacent actin molecules (59, 60). The inhibitory peptide includes residues 136–147 in cardiac TnI (cTnI) and 105–115 in skeletal TnI (sTnI). It is the interaction between this peptide and actin that inhibits CB cycling between actin and myosin. As mentioned earlier, the activation of TnC by Ca²⁺ causes TnC to "open," exposing a hydrophobic patch on the surface of the NH₂-terminal domain. It is thought that this hydrophobic patch strengthens the interaction between TnC and a region of TnI known as the "switch" region (residues 147–163), which is adjacent to the inhibitory peptide. This increased interaction pulls TnI away from its inhibitory position on the actin filament (35). This change in the position of TnI allows for tropomyosin (TM) to "roll" across the surface of the actin filament moving from a position near the outer edge of the filament (24). In its initial position on the edge of the thin filament, TM is blocking weak and strong myosin binding sites. There are a number of weak binding sites exposed at low [Ca²⁺], allowing for some weak interactions between actin and myosin, but there is no generation of force. The initial movement of TM, in response to Ca²⁺ binding to TnC, exposes additional weak binding sites allowing for myosin head attachment (24). As weak CBs form (zero force, rapid equilibrium), additional strong myosin binding sites are exposed when TM moves further across the thin filament. The formation of strong CBs leads to the generation of force by the contractile element (24).

There are differences between sTnC and cTnC in the size of conformational change caused by Ca²⁺ activating the N-domain. With sTnC, the hydrophobic patch exposed following Ca²⁺ activation is 500 Ų in area on the surface of the NH₂ terminus (16). In contrast, the size of the hydrophobic patch exposed upon the Ca²⁺ activation of cTnC is 18 Ų (51). This hydrophobic patch increases to 162 Ų when the protein interacts with a cTnI peptide corresponding to the switch region (residues 147–163) (16, 35). These results demonstrate that sTnC is more "turned on" by Ca²⁺ than is cTnC and that the activation of cTnC is highly dependent on its interaction with cTnI. The differences in sTnC and cTnC response to Ca²⁺ binding to the N-domain are due to cTnC having only one functional Ca²⁺ binding site (site II). The binding of Ca²⁺ to site I causes a rearrangement of residue side chains that contributes to the enthalpy required to overcome the energy barrier of exposing the hydrophobic core (51).

The difference in the size of the hydrophobic patch exposed upon Ca²⁺ binding to the N-domain of cTnC and sTnC during Ca²⁺ activation influences the strength at which the Ca²⁺ signal is transferred through the thin filament. Li et al. (35), using NMR solution studies, have demonstrated that the strength of interaction between the hydrophobic patch of cNTnC (exposed upon Ca²⁺ activation) and a peptide encompassing the switch region is six times less than that between sNTnC and the corresponding sTnI peptide. As it is, as this interaction pulls TnI away from its inhibitory position on the actin thin filament, the lower strength of interaction between cTnC and cTnI during Ca²⁺ activation may make it more difficult for cTnC to "pull" cTnI away from its interaction with actin, enabling the exposure of myosin binding sites. Gillis et al. (21) have suggested that the lower strength of interaction between cTnC and cTnI is responsible, at least in part, for the cardiac contractile element being dependent on the formation of strong CBs, in addition to the Ca²⁺ activation of cTn, for full activation to occur. The increased dependence of cardiac muscle on strong CBs has been suggested to be partially responsible for the steeper length dependence of force generation in cardiac muscle compared with that in skeletal muscle (21). This is another illustration of how differences in the structure/function of cTnC and sTnC translate into differences in the physiological characteristics of the two different muscle types.

	ROLE OF TnC HOMOLOG IN REGULATING THE EFFECT OF TEMPERATURE AND pH ON MUSCLE CONTRACTILITY

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
Cardiac function and skeletal muscle function have both been shown to be affected by changes in temperature. For example, as cardiac temperature lowers, the sensitivity of the contractile element to [Ca²⁺] decreases and cardiac function becomes increasingly impaired as the maximum Ca²⁺-activated force (C_max) decreases (5, 26, 28, 58). Figure 2 summarizes studies looking at the effect of temperature on the Ca²⁺ sensitivity of force generation by chemically skinned cardiac myofibrils dissected from trout, frog, rabbit, and rat hearts. In hearts from all species as temperatures decrease, the [Ca²⁺] required to generate an equal amount of force increases. Comparatively, when mammalian skeletal muscle is cooled, Ca²⁺ sensitivity increases while C_max decreases (23, 56). The difference in the influence of temperature on the Ca²⁺ sensitivity of cardiac and skeletal muscle is due, in part, to the TnC paralog expressed, while the decrease in Ca²⁺ activated force that occurs is due to a reduction in the Ca²⁺ affinity of cTnC (cardiac muscle) and a decrease in the maximal velocity (V_max) of actomyosin ATPase (cardiac and skeletal muscle). Harrison and Bers (27) demonstrated that the effect of low temperature on the Ca²⁺ sensitivity of skinned rat ventricular trabeculae was relieved when native cTnC was replaced with rabbit sTnC. This result implies that the activation of cTnC is impaired by either a reduced affinity for Ca²⁺ or changes in its interaction with cTnI required for the protein to fully activate. Recent studies have demonstrated that as temperature was reduced from 37°C to 21°C to 7°C the Ca²⁺ affinity of cTnC_Human decreased (20). Ca²⁺ affinity was measured by monitoring a fluorescent probe [Tyr inserted for Phe at residue 27 (F27W)] engineered into the protein that reports on conformational change as the protein is activated by Ca²⁺. The results of the above study suggest, therefore, that the desensitizing effect of low temperature on cardiac function is due, in some measure, to the effect of temperature on the Ca²⁺ affinity of cTnC.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Comparison of the Ca2+ sensitivity of force generation by skinned ventricular fibers isolated from hearts of: trout, frog, rat, and rabbit over a range of temperatures, while pH was maintained at 7.0. Ca2+ sensitivity was measured as the pCa at KF1/2, which is the Ca2+ concentration required to generate half-maximum force. When compared at the same temperature, the trout preparations required 10-fold less Ca2+ to generate the same measure of force than those from the mammalian species. Figure is adapted with permission from Churcott et al. (7).

	SPECIES-SPECIFIC cTnC HOMOLOGS REGULATE Ca2+ SENSITIVITY OF FORCE GENERATION

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
Living in temperate waters, ectothermic species such as the rainbow trout, Oncorhynchus mykiss, have variable body temperatures that, compared with mammalian species, are very low (5–15°C). In fact, these temperatures would be cardioplegic for a mammalian heart. One mechanism that allows for cardiac function in trout at low temperatures is that the Ca²⁺ sensitivity of the contractile element is significantly higher (>10-fold) than that of a mammalian heart when compared at the same temperature (7) (Fig. 2). This increased Ca²⁺ sensitivity is due, at least in part, to site II of cTnC_Trout having greater than twice the Ca²⁺ affinity than that of cTnC_Human, when measured at the same temperature (20). The difference in Ca²⁺ affinity between cTnC_Human and cTnC_Trout was demonstrated over a range of temperatures and pHs (7°C, pH 7.0; 7°C, pH 7.6; 21°C, pH 7.0; 21°C, pH 7.3, 37°C pH 7.0) (20). The higher Ca²⁺ affinity of cTnC_Trout allows for cardiac contraction to be initiated at lower Ca²⁺ concentrations in the trout heart compared with the mammalian heart. However, extrapolation of the data so that the Ca²⁺ sensitivity of the tissues could be compared at each of the animal's respective physiological temperature reveals that there is relative conservation of Ca²⁺ sensitivity. For example, the Ca²⁺ sensitivity of the trout heart at 7°C would be similar to the rat heart at 37°C. This idea integrates well with the concept of K_m conservation first suggested by Somero and coworkers (14, 15, 30, 54) through their extensive study of A₄-LDH from a variety of different ectothermic species with different physiological temperatures.

Through a series of Ca²⁺ binding studies using cTnC_Human mutants and cTnC_Trout mutants containing a Trp at residue 27, it was demonstrated that the residues responsible for the comparatively high Ca²⁺ affinity of cTnC_Trout are Asn², Ile²⁸, Gln²⁹, and Asp³⁰ (NIQD) (19). These four residues in cTnC_Human, in order, are: Asp, Val, Lys, and Gly and represent four of the five sequence differences between cTnC_Trout and cTnC_Human in the NH₂-terminal domain (Fig. 3). The insertion of these residues into F27W cTnC_Human increased its Ca²⁺ affinity to that of F27W cTnC_Trout (twofold that of cTnC_Human) (Fig. 4) (19). None of the identified residues are located within site II of the protein so it is through an allosteric effect that the Ca²⁺ affinity of this site is increased. In fact, there is complete sequence conservation of the residues in site II between cTnC_Trout and cTnC_Human (44).

View larger version (26K): [in this window] [in a new window]   Fig. 3. Comparison of the NH2 terminus of all known cTnC homologs. Residues identified as being responsible for the comparatively high Ca2+ affinity of cTnC_Trout are indicated in bold. Sequences are organized to indicate if they were cloned from ectothermic or endothermic species. HCM Mutant, hypertrophic cardiomyopathy mutant found in a human patient. The X in place of an amino acid indicates that the residue is not known. This is due to the sequences being cloned from DNA using trout primers.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Comparison of the Ca2+ affinity of the cTnC mutants F27W cTnC_Human (n = 10), F27W cTnC_Trout (n = 10), F27W IQD cTnC_Human (n = 10), and F27W NIQD cTnC_Human (n = 10). Ca2+ affinity is shown as the K1/2, which is the Ca2+ concentration (in µM) required to half-saturate the molecule. All values are shown as means ± SE. Bars indicated with the same letter are not significantly different from each other (P > 0.05). Measurements were made at 21°C, pH 7.0. Figure is adapted with permission from Gillis et al. (19).

View larger version (11K): [in this window] [in a new window]   Fig. 5. Ca2+ titration of steady-state force generation in single, skinned, rabbit cardiac myocytes containing either endogenous (pre-extracted) RcTnC (n = 19), WT cTnC_Human (n = 12), or NIQD cTnC_Human (n = 7) at 15°C, pH 7.0. Data are normalized with respect to the maximal force generated during each Ca2+ titration and presented as means ± SE. The curves generated by fitting the data with the Hill equation have been added to the figures for comparison against the data points. The Ca2+ concentration required to generate half-maximum force (KF1/2) are shown as means ± SE. Means indicated with the same superscript are not significantly different from each other (P > 0.05). Measurements were made at 21°C, pH 7.0. Figure is adapted with permission from Gillis et al. (19).

	STRUCTURAL CONSEQUENCES OF THE NH2-TERMINAL SEQUENCE DIFFERENCES BETWEEN TnC_TROUT AND cTnC_HUMAN

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

HNH

By monitoring [N¹⁵]-labeled F27W cNTnC_Trout during Ca²⁺ titration using 2-D{¹H,¹⁵N}-HSQC NMR, investigators have demonstrated that the residues within site I and II undergo a large positional change within the molecule during Ca²⁺ activation (4, 18, 19). A similar change occurs in the same residues in cNTnC_Human during Ca²⁺ activation. Interestingly, Ile²⁸, Gln²⁹, and Asp³⁰ are located in site I of cTnC_Trout, and Gln²⁹ in cTnC_Trout is a Leu in cTnC_Human. Gln is a hydrophilic residue, while Leu is hydrophobic. Tikunova and Davis (61) have demonstrated that the replacement of hydrophobic residues at positions 20, 44, 45, 48, and 81 with Gln in McTnC increased the Ca²⁺ affinity of all mutants. These authors suggest that the insertion of Gln decreased internal hydrophobic interactions, facilitating the opening of the molecule during activation. However, as demonstrated by Blumenschein et al. (4), residue 29 is on the surface of the molecule and, therefore, exposed to solvent. The presence of a hydrophilic residue (Gln) in cTnC_Trout at residue 29 instead of a hydrophobic residue (Leu), will not, therefore, affect the stability of the core of the molecule. It is possible, however, that the L29Q substitution could increase the interaction between the protein and its solvent, thereby decreasing local protein stability. Previous studies have suggested that such a phenomenon enables cold-adapted trypsin, -amylase, and subtilisin to function at low temperatures as a result of an increase in surface hydrophilicity, compared with warm-adapted orthologs (17, 52). By increasing the surface hydrophilicity of cTnC_ Trout, the presence of Gln at position 29 may act to destabilize this region of the protein that undergoes a large positional change during Ca²⁺ activation (19). Compared with cTnC_Human this difference could alter the free energy landscape associated with activation and help decrease the energy barrier (G) needed to be overcome for a conformational change to occur. This could, therefore, make it easier for the molecule to be activated once Ca²⁺ binds to site II.

A reduction in temperature from 30 to 7°C causes a change in the structure of cNTnC_Trout as a number of the helices shift their relative position. This change in the fold of cNTnC_Trout makes it more similar to cNTnC_Human at 30°C. In fact these two structures (cNTnC_Trout at 7°C and cNTnC_Human at 30°C) are more similar than the structures of cNTnC_Trout at 7 and 30°C. Seven degrees Celsius is within the temperature range at which cTnC_Trout normally functions, while 30°C is close to the physiological temperature of a mammalian heart. Therefore, when compared at their respective physiological temperatures, the structures of cNTnC_Trout and cNTnC_Human are very similar. In addition, functional data support this idea as the pCa₅₀ (Ca²⁺ concentration at half-maximal fluorescence), used as a measure of affinity, of cTnC_Human site II at 37°C was 5.42 ± 0.02, while this same value for cTnC_Trout at 7°C was 5.23 ± 0.03 (20). This difference in affinity is less than when the two proteins are measured at the same temperature and is comparatively minor, considering that the temperatures at which they were measured differ by 30°C. Together these results suggest that the differences in sequence between cNTnC_Trout and cNTnC_Human help to counteract the effect of temperature on protein structure and therefore allow what could be considered a similar "functional" conformation at two different temperatures.

	COMPARISON OF TnC SEQUENCES ACROSS TAXONOMIC GROUPS

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
As discussed earlier, cTnC and sTnC have different functional properties, and these differences in protein function translate into a number of functional differences between cardiac and skeletal muscle. To examine how the genes of TnC have evolved and to frame this evolution in a functional context, phylogenetic analysis on the amino acid sequences of all known TnC homologs has been completed. Ota and Saitou (45) have previously constructed phylogenetic trees using TnC as well as five other muscle protein genes cloned from a variety of species. However, the present work benefits from the increased number of TnC genes now sequenced, compared with that in the Ota and Saitou study (61 vs. 26), from a more diverse range of species (41 vs. 16) including lamprey, amphioxus, and sea squirt (Table 1). The current analysis has been completed using sTnC orthologs from 16 vertebrate species, cTnC orthologs from 25 vertebrate species, and TnC orthologs from 20 invertebrate species. Sequence and phylogenetic analyses were performed essentially as described (38). All sequences used in this study were obtained from either the National Center for Biotechnology Information (Bethesda, MD) nonredundant (nr) protein database, the Ensembl Genome Browser (Wellcome Trust Genome Campus, Hinxton, Cambridge, UK) (3), or from Ref. 62. We ensured that other members of the EF-hand Ca²⁺-binding family (e.g., calmodulin) were identified in our search, providing further confidence that no TnC sequences were missed. A complete list of all the TnC sequences used in this study is shown in Table 1 (see Supplement for FASTA amino acid sequences).¹

Phylogenetic analyses were performed using ClustalX (primarily for initial tree constructions and manipulations) and the PHYLIP package (data not shown) for verification of the initially derived trees (version 3.6b; Joe Felsenstein, Department of Genome Sciences, University of Washington, Seattle, WA) (13). Included in the alignment was the out-group calmodulin 3 sequenced from the rat, Rattus norvegicus (GenBank GI number 6978591).

Neighbor-joining trees (50) were generated using ClustalX, followed by tree evaluation with bootstrap resampling (1,000 replicates). Further phylogenetic analyses were performed using the PHYLIP package, where trees were also constructed using maximum parsimony and maximum likelihood methods (data not shown). The program TREEVIEW (version 1.6.6) (46) was used to examine and display all trees.

Alignment of all TnC, cTnC, and sTnC sequences used in the analysis reveals that TnC is conserved across a phylogenetically diverse group of organisms. A subset of these sequences, to be used as representative sequences, is displayed in Fig. 6. The high sequence identity attests to the importance of TnC in muscle contraction in both invertebrate and vertebrate species. As previously reported by Ota and Saitou (45), the tree topology reveals a clear delineation between invertebrate and vertebrate TnC homologs (Fig. 7). Invertebrate TnC homologs group together, but the lengths of the branches that link these are relatively long, representing the higher variability of the sequences within this group. Vertebrate TnC sequences are distinctly grouped into two clades representing cTnC and sTnC paralogs. Based on our analysis, the vertebrate TnC paralogs are the result of a gene duplication event specific to the vertebrate lineage. This duplication occurred sometime after the divergence of Urochordata (e.g., sea squirt) but before the divergence of Agnatha (e.g., lamprey) as lampreys have both cTnC and sTnC paralogs. It is hypothesized that such whole genome duplication occurred at some point between 500 and 600 million years ago (MYA) just before the Cambrian explosion (41). Based on their analysis of TnC, Ota and Saitou (45) suggested that fast skeletal muscle and cardiac/slow muscle became distinct after a gene duplication that occurred before the frog/mammal divergence 350 MYA. As mentioned above, one difference between the current analysis and that of Ota and Saitou (45) is that our analysis contained sequences from representative Urochordates (e.g., sea squirt) and Agnathans (e.g., lamprey). These sequences, in particular, have provided additional strength to our analysis as it is commonly thought that the vertebrate heart, as a distinct organ, first appeared in the Urochordates. Through evolution the heart has developed from a muscular tube, found in Urochordates, into the four chambered organ with coronary circulation found in higher vertebrates. For a complete review of this area please see Burggren et al. (6). One group of animals that would provide insight into timing of the evolution of sTnC and cTnC are the hagfishes, as these animals evolved after the Urochordates but before the lamprey and jawed vertebrates. It is not known if this group has either: sTnC and cTnC genes, or only one TnC. Additionally, teleost fish are thought to have underwent a second gene duplication event during the Devonian 440 MYA (41), and the salmonid fishes are thought to have undergone a third duplication 25–100 MYA (42). This would have resulted in three paralogs of cTnC and sTnC. If there were selective pressure to keep these duplicates they should be retained in the genome. However, only one cTnC_Trout gene has been identified, while no attempts have been made to clone sTnC from trout. There are currently no whole genome projects for any salmonid.

View larger version (59K): [in this window] [in a new window]   Fig. 6. Sequence comparison of representative TnC sequences. Sequences are organized into cardiac homologs, skeletal homologs, and invertebrate striated muscle homologs with representative species being listed for each. Shading indicates 4 levels of sequence conservation with black representing 90%, dark gray 70%, and light gray 50% identity. The Ca2+-coordinating positions in each EF-hand site are shown above the sequences, and the helices are labeled. Calmodulin 3 from rat is included as it was used as the out-group to root the tree. *Position of 4 residues determined to be responsible for the high Ca2+ affinity of cTnC_Trout.

View larger version (11K): [in this window] [in a new window]   Fig. 7. A maximum parsimony (MP) consensus tree of the TnC family. We included 27 representative sequences in this analysis. The MP tree was constructed by resampling 100 datasets (bootstrap) with randomized input order of 10 jumbles using Seqboot, Protpars, and Consense programs in the PHYLIP software. The tree is rooted with an out-group of calmodulin 3 (Rattus norvegicus) that is related to the TnC family. The amphioxus and sea squirt sequences fall according to their evolutionary position between invertebrate and vertebrate species; they are not considered to be orthologous to any of the vertebrate paralogs. Clear splits are observed between invertebrate striated muscle TnC and TnC paralogs (sTnC and cTnC) from vertebrate species, as well as between sTnC and cTnC at the site of TnC duplication in vertebrates, and this is indicated on the figure. Numbers indicate bootstrap values and represent the number of times out of 1,000 replicates that the same result was achieved for the respective partition.

	SEQUENCE DIFFERENCES INFLUENCING PROTEIN FUNCTION

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

Compared to site II, the sequence of site I of cTnC paralogs display lower identity and there is significant variation in the residues that compose the Ca²⁺ binding moiety of this site (Fig. 6). For example, it is the insertion of Val²⁸ and the replacement of Asp²⁹ and Asp³¹ in all cTnCs that have disrupted the ability of site I to bind Ca²⁺ (55). In addition, there is an Asp at residue 33, the "z" residue in site I, in all cTnCs, as well as in TnC cloned from the tunicate but not in any sTnCs at this position (Fig. 6). The presence of the Asp in TnC_Tunicate, as is found in cTnC, is interesting because the gene duplication that likely gave rise to a cardiac-specific TnC is thought to have occurred after the evolution of tunicates.

The differences in amino acid sequence between cTnC orthologs in site I not only alter the function of site I but also the Ca²⁺ binding characteristics of site II. For example, it has been previously established that Asn², Ile²⁸ Gln²⁹, Asp³⁰ in cTnC_Trout are responsible for the comparatively high Ca²⁺ affinity of site II. These residues are found, to some degree, in all known cTnCs cloned from ectothermic species. The equivalent residues in cTnC_Human, Asp², Val²⁸, Leu²⁹, and Gly³⁰, are present in all cTnCs cloned from endothermic species. The only exception is a cTnC_Human mutant cloned from a human heart exhibiting the pathology familial hypertrophic cardiomyopathy (FHC). FHC is a disease that can be caused by mutations to a number (>10) of genes that encode for sarcomeric proteins (49). The common phenotype of this disease is an asymmetrical enlargement of the left and/or right ventricle as well as fibrosis (49). There is no known hemodynamic etiology for this pathology. One theory is that the mutations to the different sarcomeric proteins alter the ability of the heart to be activated by Ca²⁺, thus inducing compensatory changes (49).

In the HCM cTnC_Human mutant, as in cTnC_Trout, the residue at position 29 is a Gln instead of a Leu (L29Q) (Fig. 3) (2). Liang et al. (36) have demonstrated that the Ca²⁺ affinity of the L29Q cTnC_Human mutant is intermediate between cTnC_Trout and cTnC_Human and that, when incorporated into cardiac myocytes using cTnC extraction/replacement methods, the Ca²⁺ sensitivity of force generation by the mouse cardiac myocytes is also intermediate to one containing native cTnC and a cTnC_Human mutant with the same Ca²⁺ affinity of cTnC_Trout. Using artificial Ca²⁺ transients to estimate the rate of Ca²⁺ association (k_on) to site II of F27W mutants of cTnC_Trout, L29Q cTnC_Human, NIQD cTnC_Human, and cTnC_Human, these authors suggest that L29Q and NIQD significantly increase the Ca²⁺ association rates relative to cTnC_Human when in solution (36). The authors suggest that the faster k_on is caused by the mutation destabilizing the unbound or "apo" structures on the NH₂-terminal domain, thereby making it easier for the helices to move upon Ca²⁺ activation. These results support our interpretation of data from experiments in which the structure and Ca²⁺ activated structural transition of cNTnC_Trout and cNTnC_Human were characterized by one-dimensional 1H and two-dimensional {1H,15N}-HSQC NMR spectroscopy (4, 18, 19).

A second potentially interesting residue within site I of TnC is at position 38. In all mammalian sTnCs there is a Val at this position, while in all other vertebrate sTnCs, all cTnCs as well as calmodulin, there is a Thr. The insertion of a nonpolar residue (Val) for a polar residue (Thr) may have significant effects on the functional characteristics of this site that actively binds Ca²⁺ in vertebrate sTnC. To date there have been no studies that have examined the functional consequences of this sequence difference; however, on the basis of sequence comparisons, a resulting phenotypic difference could be predicted.

There is much less sequence conservation within site I when invertebrate TnC is examined (Fig. 6). This is not too surprising as previous studies suggest that there is variation between orthologs in which of the four Ca²⁺ binding sites are functional. For example, sites I and II do not bind Ca²⁺ in TnC cloned from scallop, Chlamys nipponensis akazara, and squid, Todarodes pacificus, (10), while sites I and III are nonfunctional in TnC cloned from crayfish, Astacus leptodatylus, and barnacle, Balanus nubili, (8, 32). This demonstrates that there is much more functional and, therefore, sequence variation between these TnC orthologs.

	THERMAL STRATEGIES INFLUENCING cTnC SEQUENCE

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
Within the cTnC clade, lamprey cTnC is separated from teleost cTnCs, and these are distinctly separated from mammalian and avian cTnCs (Fig. 7). Comparison of cTnC from mammalian species reveals that there is almost complete sequence identity among homologs (Fig. 6). cTnC_Bovine, cTnC_Porcine, and cTnC_Rabbit are identical in sequence, while these proteins are 99.4% identical to cTnC_Human and cTnC_Mouse. This conservation of sequence is higher than that for either cTnI or cTnT cloned from these same animal species as sequence identity between these homologs averages 92% for cTnI and 84% for cTnT. The high sequence identity between mammalian cTnCs suggests that there are rigid structure-functional requirements for cTnC operating within the mammalian heart. Similar requirements also appear to exist in the avian heart as cTnCs cloned from the chicken, Gallus gallus, and the common quail, Coturnix coturnix, have a high degree of sequence identity to cTnC_Human (96.8–99.4%). The low sequence variability between cTnCs from mammalian and avian hearts suggests that there has been a relatively high selective pressure on this protein isoform. The high sequence identity between cTnCs from birds and mammals is remarkable considering that these two groups of animals have had separate evolutionary histories since the Carboniferous (340 MYA) (12). The negative aspect of such adaptation is that any change in these conditions, such as a decrease in temperature, could have a serious affect on function.

When cTnCs from endothermic species are compared with those from ectothermic species sequence identity between homologs begins to decrease, and this is reflected in the increased length of the branches between these two groups (Fig. 7). For example, comparison of cTnC_Human to cTnC_ Trout reveals 92% identity. There is also higher variability between cTnCs from fish and frog species compared with between mammalian homologs. For example cTnC_Trout is 94% identical to cTnC cloned from the Antarctic icefish, Chaenocephalus aceratus. This higher sequence variation likely reflects the different physiological conditions of each of the different ectothermic species from which the protein has been cloned.

Residue 30 is a Gly in all mammalian and avian cTnCs but is an Asp in all known ectothermic cTnCs (teleosts, amphibians). As Gly is a small, nonpolar residue and Asp is a hydrophilic residue, this represents a nonconservative substitution. In a previous study we replaced Asp³⁰ in F27W cNTnC_Trout with a Gly and found that the pCa₅₀ of the protein was decreased by 0.12 pCa units equal to a 32% increase in the Ca²⁺ concentration required to half saturate the molecule (22). This demonstrates that this substitution alters the Ca²⁺ activation of the protein. A second sequence manipulation, unique to cTnCs from endothermic hearts is the presence of the hydrophobic residue, Leu at residue 29. The replacement of a hydrophilic residue (Asp) with a nonpolar residue (Gly) at position 30 and the presence of a hydrophobic residue at position 29 may represent an adaptation to cTnC functioning at the relatively high (37°C) core temperature in endothermic species. These two residue replacements may act to decrease the interaction of this region of the protein with the surrounding solvent, thereby stabilizing the protein. As mentioned above, this region (site I) of the protein undergoes a significant conformational change during Ca²⁺ activation. It is likely that during vertebrate evolution, Leu²⁹ and Gly³⁰ appeared in cTnC in a common ancestor to birds and mammals and have been maintained over this evolutionary time period (340 million yr). A second, but unlikely, hypothesis is that Leu²⁹ and Gly³⁰ have arisen multiple times in endothermic species as convergent evolution. However, it is not possible to test this hypothesis as currently there are no known cTnC sequences from any reptiles that are more closely related to birds than mammals, but are ectothermic.

All cTnCs from ectothermic species, including the clawed frog, Xenopus laevis, contain at least two of the four residues identified as being responsible for the high Ca²⁺ affinity of cTnC_Trout (Fig. 3). These two are Gln²⁹ and Asp³⁰. Additionally, in cTnC from the green puffer, Tetraodon fluviatilis, there is complete sequence identity at all four positions (Asn², Ile²⁸ Gln²⁹, and Asp³⁰) (Fig. 6), while cTnC from the zebrafish, Danio rerio, birchir, Polypterus senegalus, and Fundulus heteroclitus contain three of the four residues (Fig. 3). As these identified residues, especially Gln²⁹ and Asp³⁰, are common in all ectothermic species, it is likely that their presence is not due to drift but has been selected for and therefore they may be considered to be "modifier" amino acids.

	GREATER SEQUENCE VARIABILITY IN sTnC ORTHOLOGS MAY REFLECT GREATER ADAPTIVE CHANGE IN SKELETAL MUSCLE

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
Compared to vertebrate cTnCs, there is less sequence identity in vertebrate sTnC orthologs when compared across the same range of animal species (Fig. 6). For example, there are no two mammalian homologs that have the same sequence, and there is higher variability between mammalian and avian sTnCs (90% identity = 14 sequence differences) compared with cTnCs (96.8%–99.4% = 1–6 sequence differences). The branches within the vertebrate cTnC clade are shorter than those in the vertebrate sTnC clade, indicating that the cardiac isoform has not undergone sequence divergence to the extent that the skeletal isoform has. Additionally, as mentioned earlier, comparison of human sTnC to that from E. japonicus demonstrates 70% identity compared with 83% for cTnC from these same species, equal to 48 and 31 sequence differences, respectively. The higher sequence variability in sTnC orthologs likely suggests that skeletal muscle has undergone more adaptive change than cardiac muscle. This could, as result, reflect a greater range of physiological capabilities or physiological conditions of the skeletal muscles from two species compared with the hearts of the same species.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
By considering how differences in cTnC and sTnC function relate to differences in tissue function and to the evolution of the TnC gene, we hope to provide some useful insight into how the evolution of a single protein can influence the functional capability of a tissue. By comparing the amino acid sequences of all known TnCs, we have also identified a number of residues in sTnC and cTnC that may represent interesting targets for functional studies. It is also hoped that this discussion helps to illustrate the power of a comparative approach when studying relationships between protein structure and function. Such an approach can provided unique insight into the mechanisms by which evolution has driven protein design and how these may be exploited to create novel proteins.

	GRANTS

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 
T. E. Gillis is supported by a National Sciences and Engineering Research Council (NSERC) Discovery grant. G. F. Tibbits holds operating grants from NSERC and the Heart and Stroke Foundation of BC and Yukon. G. F. Tibbits is also the recipient of a Tier I Canada Research Chair.

	FOOTNOTES

 
Address for reprint requests and other correspondence: T. E. Gillis, Dept. of Integrative Biology, Univ. of Guelph, Guelph, ON, N1G-2W1 Canada (e-mail: tgillis{at}uoguelph.ca).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

¹ The online version of this article contains supplemental material.

	REFERENCES

TOP ABSTRACT INTRODUCTION COMPARISON OF THE STRUCTURE... ROLE OF TnC HOMOLOG... SPECIES-SPECIFIC cTnC HOMOLOGS... STRUCTURAL CONSEQUENCES OF THE... COMPARISON OF TnC SEQUENCES... SEQUENCE DIFFERENCES INFLUENCING... THERMAL STRATEGIES INFLUENCING... GREATER SEQUENCE VARIABILITY IN... CONCLUSIONS GRANTS REFERENCES

 

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