In order to explore the Ca²⁺-dependent interactions between TnC and TnI, we have solved the structure of skeletal 4Ca²⁺TnC co-crystallized with a synthetic peptide, TnI[Asn96-Lys123].  Here we describe the implications of the structure for the rational design of calcium sensitizing drugs, and propose a mechanism which attempts to describe the TnC/TnI interaction in the "on" state of thin filament based regulation.
 

3.1 Overall structure description
 
 

The overall conformation of TnC in the complex is similar to that of uncomplexed 4Ca²⁺TnC [17] with an r.m.s.d. of 2.014 Å for backbone superimposition.  As in the uncomplexed structure, the long central helix is fully extended such that the N and C lobes are held separate.  Ca²⁺ is bound to each of the four EF hand domains.  The hydrophobic core of the C lobe is fully exposed to the solvent.  By contrast, that of the N lobe is shielded by residues Leu111-Asp119 of the peptide.  The rest of the peptide residues are disordered.  Helices B, C and D of TnC form a slot which constitutes the core hydrophobic interaction surface for the peptide strand (Figure 3.1).  The peptide stabilizes the conformation of the N-lobe by forming a bridge between Gln 85 and Asp86 of helix D and the BC linker (Figure 3.2).  Moreover, the sidechains of Arg113 and Arg115 form critical salt bridges with Glu54 of helix C and Asp86 of helix D, respectively.  These interactions orient the N-terminal region of the peptide as it protrudes from the hydrophobic core of the N lobe.  Peptide residues Arg112-Arg115 constitute a turn which is stabilized by the interaction of O 112 with the BC linker main chain at N 49, and by the internal van der Waals interactions between Leu111 and Val114 of the peptide.  This turn of peptide is similar in its overall conformation to that obtained in an NMR study [81]. In the current structure, it appears that the interactions of the N terminus of the peptide with the N-lobe are possibly disrupted by the strong packing interactions of helix C. There are extensive van der Waals interactions between the peptide and hydrophobic core of TnC, especially for residues Met116-Ser117 of the peptide.  These core interactions stabilize the open conformation of the N lobe in general. Together, Ile58 of helix C and the helical groove of helix D (residues Met78,  Arg81, and Gln82) comprise a small target binding pocket in EF hand 2 which is occupied by the sidechain of Met116 of the peptide.  The hydrophobic surfaces of Ser117 interact with sidechain of Met 79 on helix D and with Met43 and Leu39 of helix B .  O of Ser117 is unpaired, but it does have a close van der Waals interaction with Leu39.  The peptide strand is stabilized by the interaction between N 116 and O^e 82 of helix D.  The C terminal region of the peptide also follows the helical groove of helix D and protrudes from the TnC hydrophobic core.  The C terminal region of the peptide is stabilized by an interaction between O^d 86 of helix D and N 119.  The extensive interaction of the peptide with Asp86 is consistent with affinity measurements of the interaction of TnI segments with TnC mutant (E85A/D86A). The authors also found that the calcium dependent interaction of TnC with TnI depends on TnI residues C terminal to Met116 [90]. Moreover, TnC mutants which had deletions in this part of the central linker (Glu85-Ala87), were unable to fully activate actomyosin ATPase. Thus, it appears that these residues of the central linker and their correct interaction with TnI are critical for the transition to the troponin on state.
 

Figure 3.1
 
 

Overall structure of TnC/peptide complex
 
 

In the complex, TnC (shown in ribbon representation) has calcium ions (spheres) bound to its four EF hand domains (EF hands).  Each EF hand consists of two a-helices joined by a calcium-binding loop. The peptide (shown as ball and sticks) binds specifically to the open N lobe.  The EF hands are numbered I-IV from the N terminus.  Helices A and B are found in EF hands I. Helices C and D are found in EF hands II. Helices E and F are found in EF hands III. Helices G and H are found in EF hands IV. The N terminal helix N is disordered. (Stereo diagram generated using the program Raster3D [115].)

Figure 3.2
 
 

Peptide bound to the N lobe
 
 

The N-lobe and peptide of the TnC/peptide complex is shown. The peptide is shown as balls and sticks. The N terminal region of the peptide, Leu111-Arg115, interacts with helix C, the BC linker and helix D.  The central region of the peptide, Met116-Ser117, interacts with the hydrophobic core of the N lobe.  The C terminal region of the peptide, Ala118-Asp119, interacts with the helix D groove.  Calcium ions are shown as spheres bound to the loops of EF hands 1 and 2. (Image generated using the program POVscript [116].)
 

3.2 Comparison to other CAM superfamily members
 
 

The N-lobe of uncomplexed 4Ca²⁺TnC [17] is more open than that of the current peptide bound structure.  It appears that helices B and D clamp upon the peptide, because it is narrow, non-helical and extended in its conformation (Table 3.1).

Table 3.1

Torsional character of peptide backbone angles
 
 

LEU 111   turn
ARG 112   turn
ARG 113   turn
VAL 114   turn
ARG 115   turn
MET 116   strand
SER 117   strand
ALA 118   link
ASP 119
 
 

This TnI-dependent conformational difference was also observed in a recently published NMR structure which showed the conformation of the N lobe, but not the interactions of the peptide [46].  In contrast to the crystal structure of skeletal TnC complexed with peptide TnI(Gly1-His47) [43], the hydrophobic residues of the peptide in the current structure do not insert deeply into the core at the center of the lobe, and a small cavity is found there, enclosed by Met116 and Ser117 of the peptide, and by Met78, Ile58, Leu39 and Met43 of TnC. Interestingly, there is a similar cavity in the N lobe hydrophobic core of CAM bound to the myosin light chain kinase peptide [48], which is another case were a target peptide does not insert deeply into the lobe.

For TnC, the hydrophobic faces of the lobes do not turn inward in the current structure, nor in that of TnC bound to peptide TnI(Gly1-His47) [43], but the structures of CAM bound to various targets revealed a more compact conformation with both lobes turned inward toward the target peptide [103,48,52,104].  It is likely that this difference of interaction is related to the distinctive functions of the two proteins.  CAM is found in solution in the cellular milieu and has a variety of targets, whereas TnC remains anchored to its sole target, TnI.  TnC has a single regulatory lobe, but both lobes of CAM are regulatory.  On the other hand, CAM might also be found in an extended conformation under particular conditions where one lobe remains anchored to the target at all times. For example, CAM remains bound to phosphorylase B kinase g chain even at low Ca²⁺ concentrations [94].  CAM is expected to adopt an extended conformation when bound to the phosphorylase B kinase g chain, which shares some sequence similarity with TnI in the CAM binding regions [94].  The mechanism of this Ca²⁺independent interaction is not known at this time, but it is likely that the CAM C lobe, which does not close fully at low calcium [54,64], remains anchored to phosphorylase B kinase g chain at all times.  For TnC, the Ca²⁺ independent interaction with TnI(Gly1-His47) depends on the high affinity cation binding domains of the C-lobe, which open in response to either Mg²⁺ or Ca²⁺ binding.  Thus, the C-lobes of both TnC and CAM (in this particular case) exhibit Ca²⁺ independent interaction with their respective targets, although the mechanism of this Ca²⁺ independent interaction is likely to be different for CAM and TnC. Moreover, because of the sequence similarity in the target regions, the current structure may be representative of the interaction between CAM and phosphorylase B kinase g chain.

 

3.3 Stabilization of the central linker by peptide interactions
 

3.3.1 Packing considerations

One of the most striking features of the current structure is the stabilization of the helical central linker by the peptide (Figure 3.3).  Spin labeling studies have shown that the binding of cTnI(86-211) to cTnC decreases the flexibility of the central linker and maintains cTnC in an extended conformation [89]. This is consistent with x-ray and neutron scattering results which showed that, in the presence of Ca²⁺, TnC assumes an extended conformation in its interaction with TnI, with a maximum linear dimension of about 72 Å [91].  By contrast, the 2Ca²⁺TnC/TnI(Gly1-His47) complex [43] has a maximum linear dimension of less than 60 Å.  In the current structure, helix D has packing interactions with loop 4 of the adjacent protomer.  This protomeric loop 4 also provides the central linker with its only packing interaction:  The sidechain of Lys88 of the central linker interacts with the sidechains of Asp147 and Asp149 of the adjacent protomer.  The central linker is otherwise fully exposed to the large solvent channels.  In fact, the central linker forms a helical pillar which holds apart the layers of lobes in the xz planes of the triclinic crystal so that the relative disposition of the independent layers is determined by the disposition of the central linker (Figure 3.4).  Thus, it appears that the stability the crystal depends on the stability of the central linker in its extended helical conformation.
 

Figure 3.3
 
 

A comparison of the central linker stabilization in various crystal structures of TnC
 
 

In the current structure (top left) the N terminal portion of central linker is constrained by its interaction with the peptide (Rabbit skeletal numbering is used thoughout for consistency). These constraints lead to a strengthening of the intrahelical interactions of the central linker (Table 3.2). The central linker of 2Ca²⁺TnC [15] (top right) is constrained in a similar way by the BC linker and the intrahelical interactions are also strengthened. Correspondingly, the central helix has a similar disposition overall in the current structure and in that of 2Ca²⁺TnC. By contrast, the central linker of 4Ca²⁺TnC (bottom left) does not receive as much stabilization [17], and the intrahelical interactions of the linker are weakened (Table 3.2). The various crystal forms of 4Ca²⁺TnC have dissimilar bends in the central helix [112]. In the complex of 2Ca²⁺TnC and peptide TnI(Gly1-His47) [43] (bottom right) the central linker is unwound due to the interactions of the peptide. Although the N lobe is in the 2Ca²⁺ state, the linker stabilizing interactions of Asp86 and Glu85 with the BC linker are broken. Thus, it appears that the central linker may play a role in regulation, because its stability is coupled to the state of the N lobe.
 

Table 3.2
 
 

Intrahelical central linker interactions
 

          TnC/peptide     2Ca2+TnC     4Ca2+TnC1     4Ca2+TnC2

Oe94-Nz90(Å)           4.10                     5.12                     6.86                     8.40

Oe92-Nz88(Å)           3.33                     3.46                     3.04                     7.30

OHg91-O87(Å)          3.25*                     -                       4.77                     4.19

OHg91-O88(Å)             -                       2.85                   4.04                     5.08

3.3.2 Ca²⁺ dependent central linker flexibility

A comparison of 4Ca²⁺TnC and 2Ca²⁺TnC led to the prediction of Ca²⁺ dependent flexibility in central linker in Chapter 2.  This prediction was based not only on the crystallographic structures, but also on the results from an NMR study [57] and on the structures of other members of the CAM superfamily [48,58,60,67,68].  These additional studies indicated the precise regions of unwinding in the central linkers of various members of the CAM superfamily. Based on this comparison, and on Ca²⁺ dependent interaction of the BC linker with the central linker, also described in Chapter 2, it appeared that the central linker would be more flexible in the 4Ca²⁺ on state than in the 2Ca²⁺ off state.  Moreover, a Ca²⁺ dependent unwinding of the central linker at residues Lys84-Asp86 was predicted. Although this prediction received some support from the other structures, it seemed at the time to border on dangerous speculation.  Perhaps we were unaware that NMR studies had shown a Ca²⁺ dependence of central linker flexibility [89].  This prediction has received additional experimental support in the three years since the initial publication of Chapter 2 in Structure [17].  For example, a comparison of more recent crystallographic structures of 4Ca²⁺TnC reveals general variability in the disposition of the central helix (although the linker is still fully helical) [112], in contrast to that of 2Ca²⁺TnC.
 

Figure 3.4
 
 

The central helix supports layers of lobes in the crystals
 
 

The C2 dimer forms the asymmetric unit of the P1 cell.  Two-fold symmetry is still present in the individual yz planes of the P1 crystals shown here.  The central helix forms a helical pillar which holds apart the layers of lobes in the xz planes of the P1 crystal form.  In fact, the relative disposition of the xz planes depends on the stability of the central linker, because no other interactions bridge the layers.  Thus, it appears that the stability of the crystals depends on the integrity of the helical central linker in the TnC/peptide complex. (Figure generated using the program RASMOL [117])
 
 

In the structure of 2Ca²⁺TnC bound to peptide TnI(Gly1-His47) (Figure 1.2) [43], the central linker is unwound and the overall conformation of the complex is relatively compact, because the peptide interacts with both the N and C lobes.  Similarly, the structure of the bepridil-4Ca²⁺⁺cTnC complex, also shows that the central linker unwinds when there are interactions that hold both lobes together [114].  By contrast, the N-lobe of the bepridil-4Ca²⁺⁺cTnC complex is open, but the N-lobe of 2Ca²⁺TnC bound to peptide TnI(Gly1-His47) is closed, so that the central linker receives some stabilization from van der Waals interactions with the BC linker and helix B.  In accordance with the prediction, N terminal central linker residues Lys84-Glu85 are helical, and Asp86 forms single helical interaction. In this case, Asp86 has a relatively high B factor, because it is destabilized by the adjacent linker region which is unwound.  In the bepridil-4Ca²⁺⁺cTnC complex, these residues are unwound in accordance with the prediction. This Ca²⁺⁺ dependent flexibility points to a possible structural role for the central linker of TnC in thin filament regulation.
 

3.3.3 Intrahelical stabilization of the central linker

Ever since the crystal structure of 2Ca²⁺TnC was first shown to contain a fully helical central linker [96, 97], the possibility of internal stabilization within the central helix has been discussed [98].  If the peptide stabilizes the helical conformation of the central linker, then it could be expected that these internal stabilizing interactions would also be strengthened due to the helical dipole [17].  In the N lobe of 2Ca²⁺TnC [15], these central linker stabilizing interactions of the peptide are mimicked by the BC linker of TnC (Figure 3.3).  Thus, it would be expected that internal stabilization of the helical central linker would also be observed in the 2Ca²⁺TnC crystal structure.  By contrast, the intrahelical stabilizing interactions of the central linker would be weaker in the crystal structure of 4Ca²⁺TnC, where the central helix receives less stabilization [17].  Moreover, central linker stabilization by the BC linker would be weaker in cases where the central linker is non-helical, such as in solution, or in the crystal structure of 2Ca²⁺TnC bound to peptide TnI(Gly1-His47) [43].  This destabilization may facilitate the binding of the inhibitory region of TnI, because these interactions must be broken to open the lobe [17]. Table 3.2 shows that central linker intrahelical interactions are better overall for the current complex and 2Ca²⁺TnC than for 4Ca²⁺TnC.  Figure 3.3 shows that the various crystal structures are in good agreement with this notion of central linker stabilization.  It has been suggested that the phi/psi rotation of Gly89 would be a first step in the unwinding of the central linker [17].  It now appears that these intrahelical interactions provide some stabilization against unwinding at Gly89 under conditions where the central linker receives additional stabilization at Gln85 and Asp86 (Figure 3.3).  Moreover, the various crystal structures of TnC together show that the stabilization of the central linker depends on the state of the N lobe (Figure 3.5, Table 3.2).
 
 

3.3.4 The CAM analogy

It has been suggested that increased flexibility of the central linker could improve the interaction of TnI with 4Ca²⁺TnC because, by analogy, the central linker is unwound in every known peptide bound structure of CAM [17,103,48,52].  Accordingly, TnC might interact with TnI in a less extended conformation than that observed in the 2 or 4Ca²⁺TnC crystallographic structures.  As more structures have appeared, the facts have become clearer. In the peptide bound complexes of CAM and in structure of 2Ca²⁺TnC bound to peptide TnI(Gly1-His47), the central linker is unwound because the peptide stabilizes close contacts between the lobes [43,103,48,52]. A similar situation exists for the bepridil-4Ca²⁺⁺cTnC complex [114]. By contrast, in the complex of TnC with peptide TnI(Asn96-Lys123), the peptide can stabilize a less flexible, helical central linker that holds the lobes apart.
 

Figure 3.5
 
 

A mechanism of interaction between TnC and TnI
 
 

In the proposed mechanism, step 1 is represented by the complex of 2Ca²⁺TnC and peptide TnI(Gly1-His47) [43]. TnC is not extended and the central linker is unwound. In step 2, calcium ions bind to the N lobe, and it opens and becomes receptive to interaction with the inhibitory region of TnI.  In step 3, the inhibitory region of TnI binds and stabilizes the helical conformation of the central linker and the extended conformation of the complex.

3.3.5 The central linker in solution

NMR studies find (in contrast to the crystal structures) that the central linker melts and is non-extended when TnC is isolated in solution [95,60].  (There is a similar inconsistency with respect to the interhelical angles of the crystal and NMR structures; see Chapter 2).  This troublesome fact can be explained by noting that these are solution structures, where there is greater freedom of movement.  In contrast to TnC, CAM is a solution protein, and the central linker of CAM is also known to unwind in solution [57,58]. It is notable that the central linker of CAM has also been observed as helical in crystal structures [49].  Thus it appears that the extended conformation of CAM or TnC is not stable in solution. In this sense, crystallographic structures may better reflect the structure of TnC in the native environment, because like the thin filament, crystals are protein arrays which can apparently stabilize the extended conformation of protein molecules.

3.3.6 Implications of TnI interactions with the central linker

In previous structures of uncomplexed TnC helix D is stabilized by helix N, but in the current structure, helix D and the central linker are stabilized by the peptide.   There are extensive van der Waals interactions between helix D and peptide which were described earlier.  Also, an interlocking chain of interactions between the peptide and TnC that constrain the side chains of helix D and the central linker at Gln85 and Asp86 (Figure 3.3), so that TnI residues Leu111-Asp119 provide exceptional stabilization to the central linker of TnC.

A possible implication of the current structure is that the central linker is extended and helical in the on-state of thin filament regulation.  Such an argument finds some support in experiments which suggest a role for the central linker in regulation [101,108,109,110] and in energy transfer experiments on the TnC/TnI complex in the presence of Ca²⁺ [40].  Regulation may require the added specificity implied by the many TnC/peptide interactions in the current structure.  For example, the TnI point mutation Arg113Gly has been implicated in hypertrophic cardiomyopathy (HCM) [105,106,107].  This mutation leads to an increase in Ca²⁺ sensitivity [106,113]. As noted above this arginine makes a critical salt bridge to Glu54 of helix C (Figure 3.2).  In the mutant, the steric constraints on this region of TnI are likely to be weaker, so that TnI would bind to TnC at lower Ca²⁺ levels.  It should be noted that this mutation is

also likely to affect the interaction of TnI with actin and to impair regulation [113]. The current structure only provides a probable explanation of the Ca²⁺ sensitivity effect. In another example, the mutation of Asp86 of the central linker to alanine results in reduced affinity for the peptide [41] and in defective actomyosin ATPase activation in avian TnC [111]. As described above, Asp86 makes many contacts with the peptide and is critical for the stabilization of the central linker. Thus, it is likely that the highly specific central linker stabilizing interactions in the current structure are representative of those in the troponin on state.

 

3.4 TnC/TnI interaction mechanism

As mentioned previously, the central linker has different dispositions in the current structure, and in that of 2Ca²⁺TnC bound to peptide TnI(Gly1-His47) [43].  Based on this observation, a mechanism for the calcium-dependent interaction of TnC with TnI can be proposed which employs these structures.  The first step is represented by the TnC/TnI(Gly1-His47) crystal structure [43] (Figure 3.5).  In step 2, when Ca²⁺ binds, the N lobe simply opens.  The overall conformation of TnC is still relatively compact, but the N lobe is receptive to the calcium dependent interaction with TnI (Figure 3.5).  Although this structure has not been directly observed, the N lobe interactions with peptide TnI(Gly1-His47) have been detected in the presence of Ca²⁺ [92].  In step 3, the extension of the central helix probably accompanies the binding of TnI to the N lobe for reasons explained above (Figure 3.5).  It should be noted that a fully helical central linker is not required for full extension of the central helix.  In other words, it is likely that the N and C lobes could still be held apart by their interaction with TnI, even if the central linker remains partially unwound.  Nevertheless, it now seems likely that the added stability of the central linker in the presence of TnI(Leu111-Asp119) would be exploited in the TnC/TnI complex.  In a final step, when Ca²⁺ is released from EF hands 1 and 2,  TnI is simply expelled from the closing N lobe.  If the extended conformation of TnC is no longer stabilized by TnI, the central linker would unwind and TnC would resume its step 1 conformation. The overall structural disposition of TnC in this final step is unclear at present, but if it were known, a regulatory mechanism would be implied by it.

This mechanism implies a large conformational change in TnI which would accompany the rotation and displacement of the N lobe in step 3.  There is an expected displacement of over 25Å for the loop between helices A and N with respect to loop 4 of the C lobe and the N-terminus of TnI (Figure 3.5).  In intact thin filaments, the inhibitory region of TnI would likely be held away from its actin binding site in the on state, represented in step 3.  These observations are roughly consistent with the results of energy transfer experiments which showed that Cys133 of TnI moves 15 Å away from Cys374 of actin and 12.6 Å closer to TnC on Ca²⁺ binding [99, 102].  It is tempting to suggest that this interaction mechanism represents the conformational switch of thin filament regulation, however, this suggestion is not supported by our results.  The current structure shows only the Ca²⁺ dependent interaction of the N lobe of TnC with a short region of the peptide representing TnI(Asn96-Lys123).  Although this structure cannot represent fully the TnC/TnI interaction nor its role in regulation,  nonetheless, we have shown that these interactions may represent important aspects of the regulatory role of TnC/TnI interaction in vertebrate striated muscle.

 

3.5 Implications for the design of drugs targeting TnC

It has recently been shown that the deep cavity of the N lobe hydrophobic core is the interaction site in cTnC for the phenyl ring of bepridil, the calcium sensitizing drug [114].  It was also proposed that cTnI and bepridil could bind to the cardiac N-lobe simultaneously [89].  It is likely that the complex of cTnC and cTnI resembles the current structure.  An overlay of the bepridil-bound cTnC N lobe with the current structure (Figure 3.6) does produce a clash between (Met116-Ser117) of the peptide and the isobutoxy chain of bepridil.  This clash is probably not significant because the isobutoxy chain is quite flexible.  Thus, the suggestion appears to be plausible that N lobe of cTnC can accommodate both cTnI and bepridil.  In such a case, the "wedge-like" insertion of bepridil into the N lobe would result in a more fully open N lobe, and the backbone interaction of Arg144 of cTnI (sTnI Arg112 equivalent) with the BC linker of cTnC (sTnC mainchain N 49 equivalent) would likely be broken.  To compensate, hydrogen bonds could be formed between the positively charged pyrrolidine groups of bepridil and several carbonyl oxygens of N-terminus of the peptide (Figure 3.6). Thus, an important implication of this comparison is that bepridil interacts not only with TnC but with TnI as well.  This is consistent with the observed effect of bepridil, the modulation of contraction in cardiac muscle.  Furthermore, modifications of bepridil, such as the substitution or addition of more basic amphipathic groups for the pyrrolidine group and the truncation of the isobutoxy chain, may produce a more powerful calcium sensitizer.  Additionally, cTnI and the drug bind to largely different surfaces in the N lobe, so a drug binding surface can be mapped, which takes into account the TnC/TnI interaction.  For example, the molecules "Bep1" and "Bep3" can be used to construct an adductive "model drug" (Figure 3.6), which may be a more specific calcium sensitizer, enhancing contraction.  Alternatively, bulkier compounds which obstruct the TnI binding surface may attenuate the contractile response.  Such an agent might be useful in the treatment of excessive calcium sensitivity in diseases such as hypertrophic cardiomyopathy [105,106,107].
 

Figure 3.6
 
 

An overlay showing possible interactions of cTnI with cTnC and bepridil
 
 
 

An overlay of the A and D helices was used to model the peptide of the current structure into the N lobe of cardiac TnC (medium grey) with bepridil (dark) bound.  Some possible interactions are shown with dotted lines between the carbonyl groups of the peptide and the nitrogens of bepridil.  The sidechain of Bep1 clashes with the mainchain of the peptide, but this clash is not likely to be significant because the sidechain is quite flexible, and should be displaced by the peptide (bepridil sidechain not shown).  It appears that the drug and the peptide bind to different surfaces of the cTnC N lobe, and that the bound bepridils define a drug binding surface on the N lobe.  A polycarbon linkage between Bep1 and Bep3 was used to construct the "model drug".
 

Figure 3.7
 
 

Four structures overlayed: Interactions of TnI with TnC
 
 
 

This model was produced by fitting the shared coordinates of the following structures to those of the current structure; TnI(Gly104-Arg115) [45] (blue) bound to EF hand II, TnI(Glu3-Glu33) [43] (grey) bound to EF hands III and IV, cTnI(Arg147-Lys163) [47] (red) shown with skeletal numbering and bound to EF hand I. The four EF hands of TnC are numbered with roman numerals and TnI(Leu111-Asp119) (yellow) is shown as in the current structure, bound to EF hands I and II. The shared regions of the overlayed structures are not shown. In the model residues TnI(Lys105-Lys107) make plausible interactions with EF hand II of TnC in accordance with crosslinking studies [31,32,118]. These interactions do not appear in the current structure due to packing constraints around helix C. Residues TnI(Arg115-Ala118, skeletal TnI numbering) of the cardiac TnC/TnI(Arg147-Lys163) NMR structure [47] are not shown due to a clash with the current structure in the core binding region. This clash is probably due to the fact that these residues are not stabilized by cTnC(Asp86, rabbit skeletal numbering) in the NMR structure because the central linker is truncated and unwound [47].
 
 
 

3.6 Materials and Methods

We have determined to 2.0 Å resolution the crystal structure of expressed rabbit 4Ca²⁺TnC grown in the presence of a synthetic peptide comprising TnI residues Asn96-Lys123. Two crystal forms have been obtained: monoclinic (C2) with unit cell dimensions a=109.40 Å, b=26.54 Å, c=59.67 Å, b= 93.44° and triclinic (P1) with unit cell dimensions a=26.28 Å, b=55.29 Å, c=63.26 Å, a=89.53° b=91.46° g=103.72° Crystals of both forms were grown by seeding under identical conditions at 4° C from a mixture of 2 µl of reservoir buffer (50 mM cacodylate pH 6.5, 10 mM CaCl2, 29% ethanol, 29% MPD). The partial specific volume is similar for both crystal forms (~2.8 Å3/Da), with one and two molecules per asymmetric unit for the C2 and P1 crystal forms respectively. Data were collected from the monoclinic crystal form at -180° C to 3.5 Å resolution using a MAR image plate detector mounted on a Rigaku X-ray generator equipped with Supper focusing mirrors. Data for the triclinic crystal form were collected to a resolution of 2.0 Å at -160° C using synchrotron radiation at CHESS (beamline A1,l =0.935 Å). Data reduction was carried out with the programs DENZO [77] and SCALEPACK [78] Table 3.3 summarizes the statistics of diffraction.

The program AMoRe [85] was used to orient and position the N and C lobes of 4Ca²⁺TnC independently in the monoclinic asymmetric unit. The final correlation coefficient after fitting was 44.4 (R factor 47.4) for all the data between 8 and 3.5 Å. After 400 cycles of refinement with the program XPLOR [81] the R factor fell to 0.349. A comparison of the unit cell parameters for the monoclinic and triclinic crystal forms suggested that the C2 TnC dimer could be used as a search model in AMoRe for the P1 cell. The final correlation coefficient after fitting was 48.8 (R factor 46.2). After one round of simulated annealing with NCS restraints, the R factor fell to 0.330. F0-Fc maps indicated peptide binding specifically to the N lobe but not to the C lobe. The program O [83] was used to model the peptide into this density and to improve the fitting of the TnC molecules to the electron density map (Figure 3.8). The solvent structure was modeled automatically with the program ARP [82].  The NCS restraints were removed in a final round of maximum likelihood refinement with the program CNS [93].  The P1 protomers in the final structure are nearly identical with an rms displacement of 0.140 Å and 0.076 Å for the protein and peptide respectively.  Table 3.4 gives the refinement statistics for both crystal forms.
 

Figure 3.8
 
 

Electron density map for TnI[Leu111-Asp119]
 
 
 

The final peptide map was calculated using the program DM [79] with non-crystallographic symmetry averaging.

 

Table 3.3

Data collection statistics

Crystal form                monoclinic        triclinic

Resolution range (Å)        15.0 - 3.50       50.0 - 2.00
No. of reflections          6573              193252
No. unique reflections      2231              21185
Completeness                97.08             90.9
Rmerge (%)                  8.8               7.1
 
 
 

Table 3.4

Refinement statistics

                                    monoclinic        triclinic

Number of reflections               2225              21183
Sigma cutoff                        none              none
R factor (%)                        29.6              21.8
Free R Factor (%)                   33.4              25.8
Number of water molecules           0                 131
Rms bond lengths (Å)                0.035             0.008
Rms bond angles (°)                 2.744             1.436
Rms bond dihedrals (°)              25.284            20.919
Rms bond impropers (°)              4.085             0.832
Global G factor                     -1.0              0.4
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Before we commenced this work, the only TnC structure that was known was that of 2Ca²⁺TnC [15,16]. With its closed N lobe, this structure represents the off state. Based on this structure, and on a comparison to CAM, Herzberg, et al. proposed that the N lobe would open in the presence of Ca²⁺[22]. It was impossible to describe the structural role of TnC in thin filament regulation based on this one structure, so we first set out to crystallize TnC in the 4Ca²⁺, on state. The structure of 4Ca²⁺TnC validated the proposal of Herzberg et al. [22], and revealed at atomic resolution the hinge regions and critical linkages that change in the transition from closed to open. We were also able to show how sequence differences give rise to many of the diverse functional properties of proteins in the CAM superfamily.

Another insight derived from the structure of 4Ca²⁺TnC was the prediction of Ca²⁺ dependent flexibility in the central linker, which arises from TnC specific interactions between the BC linker and the central linker in the 2Ca²⁺ state. In light of the work described in Chapter 3, this now appears as a critical insight. Recently, the structure of the bepridil-4Ca²⁺⁺cTnC complex has been solved [114]. In the structure, both lobes of cTnC are open, and the central linker is unwound.  We have shown in Chapter 3 that a comparison of the bepridil bound structure to that of 2Ca²⁺TnC bound to peptide TnI(Gly1-His47) [43] provides the first unambiguous crystallographic support for the predicted Ca²⁺⁺ dependent flexibility in the central linker.

In Chapter 3, the analysis of the interaction between 4Ca²⁺TnC and TnI(Asn96-Lys123) suggests the following interpretation of this Ca²⁺ dependent flexibility in central linker; Although the central linker of 4Ca²⁺TnC is more flexible than that of 2Ca²⁺TnC, this increase in flexibility can be reversed by the Ca²⁺ dependent interaction of TnI with 4Ca²⁺TnC. Our structure shows that TnI provides stabilization to a less flexible and helical central linker similar to that observed in 2Ca²⁺TnC (17).  In particular, TnI residues Arg115-Asp119 provide stabilization to the N terminal turn of helix in the central linker. This N terminal turn of helix (residues Lys84-Asp86 of sTnC) comprises residues known to be crucial for regulatory activation [101,41]. In Chapter 3, we have shown that the cruciality of these central linker residues is attributable to their specific interaction with TnI(Arg115-Asp119).  Moreover, because these interactions appear to be part of the regulatory switch, the stabilization in TnC of an extended helical central linker by TnI is probably an important feature of the troponin on state. We have also shown how a comparison of the current structure to that of bepridil-4Ca²⁺⁺cTnC may guide the development of new drugs that are specific to the troponin complex.
 
 

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