Calcium-dependent Changes in the Flexibility of the Regulatory Domain of Troponin C in the Troponin Complex

Abstract

With the recent advances in structure determination of the troponin complex, it becomes even more important to understand the dynamics of its components and how they are affected by the presence or absence of Ca 2؉ . We used NMR techniques to study the backbone dynamics of skeletal troponin C (TnC) in the complex. Transverse relaxation-optimized spectroscopy pulse sequences and deuteration of TnC were essential to assign most of the TnC residues in the complex. Backbone amide 15 N relaxation times were measured in the presence of Ca 2؉ or EGTA/Mg 2؉ . T 1 relaxation times could not be interpreted precisely, because for a molecule of this size, the longitudinal backbone amide 15 N relaxation rate due to chemical shift anisotropy and dipole-dipole interactions becomes too small, and other relaxation mechanisms become relevant. T 2 relaxation times were of the expected magnitude for a complex of this size, and most of the variation of T 2 times in the presence of Ca 2؉ could be explained by the anisotropy of the complex, suggesting a relatively rigid molecule. The only exception was EF-hand site III and helix F immediately after, which are more flexible than the rest of the molecule. In the presence of EGTA/Mg 2؉ , relaxation times for residues in the C-domain of TnC are very similar to values in the presence of Ca 2؉ , whereas the N-domain becomes more flexible. Taken together with the high flexibility of the linker between the two domains, we concluded that in the absence of Ca 2؉ , the N-domain of TnC moves independently from the rest of the complex.The troponin (Tn) 1 complex is responsible for the regulation of contraction in striated skeletal and cardiac muscles. Although many structural studies have been done of pairwise interactions between components of the complex (for reviews see Refs. 1-4), the details at an atomic level of how the complex works as a whole have not as yet been fully elucidated. Recent determination of the crystal structure of cardiac troponin (5) has revealed an apparently quite flexible complex. This has focused interest on the need for dynamics information on the troponin complex in order to properly interpret the structural data.The three components of Tn, troponins I, C, and T, are responsible for inhibiting muscle contraction in the absence of Ca 2ϩ , sensing the change in intracellular Ca 2ϩ levels, releasing the inhibition in the presence of Ca 2ϩ , and transmitting this information to the other components of muscle thin filaments, respectively (for a recent review see Ref.2). Troponin C (TnC) is a Ca 2ϩ -binding protein containing two domains connected by a linker. The linker is a straight ␣-helix in the crystal structure of isolated TnC (6 -8) but is flexible in solution (9). Each domain contains two EF-hand metal-binding sites, and the helices that flank those sites are named A to D in the N-domain and are named E to H in the C-domain. An extra helix at the N terminus is named N. The sites in the N-domain are Ca 2ϩ -specific and important for the regulation of muscle contract...

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

“…Although the specific structural features of this extension cannot be deduced from the low-resolution neutron models, the novel finding of this structural change in sTnI has laid a foundation for further high-resolution studies. 15,[63][64][65] All optimized SANS-derived models point to a similar shape for sTnC, that of a fully extended dumbbell shape in both Ca 21 -free and Ca 21 -bound states, consistent with previous interpretations 48,49 as opposed to the somewhat more compact conformation observed in the cTn crystal structure 60 and SANS models for cTn. 50,51 In the SANS-derived model for sTn, the N-terminal domain of Ca 21 -bound sTnC rotates more than 1008 away from the position seen in the crystal structure.…”

Structural changes in troponin in response to Ca 2+ and myosin binding to thin filaments during activation of skeletal muscle

“…This is in marked contrast to the behavior of the C helix, in the regulatory head, which tilts by 26°in direct response to Ca 2ϩ binding, and is insensitive to myosin head binding in physiological conditions. These distinct tilting motions of the regulatory head and IT arm during activation are consistent with the flexibility between the two domains inferred from in vitro structural studies (8,9,16). The multiple conformations of the IT arm observed here may be related to the ''blocked,'' ''closed,'' and ''open'' states of the thin filament described in in vitro biochemical and structural studies (29,30).…”

“…1), but in the Ca 2ϩ -free state the D/E linker is melted (9). The junction between the two domains of TnC in the core complex in solution is more mobile in the absence of Ca 2ϩ (16).…”

Switch Action of Troponin on Muscle Thin Filament as Revealed by Spin Labeling and Pulsed EPR

“…It is reasonable to resolve this discrepancy by assuming that the N-lobe of TnC is mobile and tumbling relative to the C-lobe of TnC and the actin surface (26,48), whereas the regulatory region of TnI is released from TnC and fixed on the actin surface. The flexibility of the N-lobe of TnC in the complex was reported by Blumenschein et al (49) using NMR. In the crystal structure of chicken skeletal troponin, the central helix is melted in the ϪCa 2ϩ state, whereas it is folded in the ϩCa 2ϩ state (22).…”