Interactions and Dynamics within the Troponin Complex

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1 Interactions and Dynamics within the Troponin Complex Tharin Blumenschein Steve Matthews Lab - Imperial College London (formerly Brian Sykes Lab, Canada)

2 Striated muscle

3 Thin filament proteins - regulation Troponin I - inhibits muscle contraction Troponin C - releases inhibition in the presence of calcium Troponin T - binds to the other thin filament proteins, transmitting the conformational changes Smillie et al. Gordon et al., 2000

4 Crystal structure and SANS/SAXS model of skeletal troponin Vinogradova et al. (2005) PNAS 102, 5038 (PDB: 1YTZ) King et al. (2005) J. Mol. Biol. 345, 797

5 The importance of flexibility in cardiac troponin Takeda et al. (2003) Nature 424, 35

6 What we used Chicken skeletal TnC, TnI and TnT-T2 55 kda complex [ 2 H, 13 C, 15 N]-TnC + 2 H-TnI + 2 H-TnT-T2 2 H-TnC + [ 2 H, 13 C, 15 N]-TnI + 2 H-TnT-T2 In 90% H 2 O, 10% D 2 O TROSY for assignment Relaxation in the complex

7 Skeletal TnC in the complex TnC: + TnI peptide In the complex The structures of the TnC domains in the Tn complex are very similar to the domain+tni peptide complexes.

8 Adding EGTA/Mg 2+ Chemical shift changes Mainly in the N domain N-domain of TnC becomes apo, C-domain doesn t - as expected from affinities Similar chemical shifts to apo isolated N-domain

9 Skeletal TnI in the complex The C-terminal region of TnI is much more flexible than the rest of the molecule, and moves independently from the rest of the complex. The presence or absence of Ca 2+ does not change the spectrum significantly.

10 C α Chemical Shift Index Calcium EGTA TnC residue residue Calcium EGTA Calcium EGTA residue TnI Calcium EGTA residue

11 TnC Relaxation - T 2 values are as expected Molecular weight 800 MHz Ca 2+ Isotropic, model-free HYDRONMR Measured Model-free approach: isotropic tumbling, fast internal motions, S 2 =0.85; τ m =MW/2 R 1 =(d 2 /4)[J(ω H -ω N ) + 3J(ω N ) + 6J(ω H +ω N )] + c 2 J(ω N ) 26 ms 20 ± 3 ms 24 ± 5 ms R 2 =(d 2 /8)[J(0) + J(ω H -ω N ) + 3J(ω N ) + 6J(ω H ) + 6J(ω H +ω N )] + (c 2 /6)[4J(0) + 3J(ω N )] + R ex J(ω)=S 2 τ m /(1+ω 2 τ m2 ) T 2

12 TnC T 1 relaxation times are shorter than expected R 1 = R 1DD + R 1CSA + R 1other R 1DD + R 1CSA» R 1other R 1 = R 1DD + R 1CSA Only for small proteins!! 800 MHz Ca 2+ Isotropic, model-free HYDRONMR Measured T s 3.5 ± 0.7 s 1.60 ± 0.05 s As molecular weight and τ m increase, CSA and dipole-dipole interactions become more inefficient for the longitudinal relaxation. Other mechanisms, which can be ignored for smaller molecules, start to become relatively more important (interactions with dissolved O 2, contaminant paramagnetic metals, chemical or conformational exchange).

13 Relaxation times T 2 change in the presence of EGTA/Mg MHz T2 (ms) EGTA Calcium 800 MHz N domain more mobile Central linker is very flexible Site III is flexible Val 65 very flexible EGTA Calcium residue

14 N domain of TnC is flexible in the presence of EGTA/Mg 2+ Ca 2+ EGTA N-TnC N-TnC C-TnC Li et al. (2005) J. Muscle Res. Cell Motil. 26, 1

15 N domain of TnC is flexible in the presence of EGTA/Mg 2+ Ca 2+ EGTA N-TnC N-TnC C-TnC Li et al. (2005) J. Muscle Res. Cell Motil. 26, 1

16 T 1 relaxation times for TnI MHz Calcium 800 MHz Calcium 600 MHz EGTA 800 MHz EGTA residue The structure and flexibility of the C-terminal region of TnI are not significantly affected by the presence or absence of calcium.

17 NOE ratios for TnI MHz Calcium 800 MHz Calcium 600 MHz EGTA 800 MHz EGTA -2.1 residue Very flexible region of TnI

18 T 2 relaxation times for TnI MHz Ca 600 MHz Ca 600 MHz EGTA 800 MHz EGTA residue

19 T 2 relaxation times for TnI MHz Ca 600 MHz Ca 600 MHz EGTA 800 MHz EGTA residue The gradual increase in T 2 values as we move down the sequence suggests a tethered domain.

20 Reduced spectral density analysis - J(0) MHz Calcium 800 MHz Calcium 600 MHz EGTA 800 MHz EGTA residue The gradual decrease in slow movements suggests a tethering point; the slight increase around residue 165 suggests a collapsed region.

21 Reduced spectral density analysis - J(ω N ) egta 800 egta 800 calcium 600 calcium residue J(ω N ) stays constant for most of the C-terminal region of TnI; the exception is the flexible C-terminus

22 Reduced spectral density analysis - J(0.87ω H ) egta 800 egta calcium 600 calcium residue Fast motions increase gradually along the structure.

23 C-terminal region of TnI The C-terminal region of troponin I is an independent, highly flexible domain. This domain is weakly structured, with at most nascent structure. The structure is probably stabilised upon binding to actin, in the muscle relaxed state.

24 Flexibility in the troponin complex Flexibility is critical for the functions of both TnC and TnI. Flexibility of the central linker of TnC allows the N-terminal domain to move around and reach for TnI to interact with. Flexibility in the C-terminal region of TnI is likely linked to the energetics of binding to actin.

25 Acknowledgements Brian Sykes Dave Corson Deborah Stone (Fletterick Lab - UCSF) Sykes Lab (AR45659)