Mechanically induced titin kinase activation studied by force-probe molecular dynamics simulations

Abstract

The conversion of mechanical stress into a biochemical signal in a muscle cell requires a force sensor. Titin kinase, the catalytic domain of the elastic muscle protein titin, has been suggested as a candidate. Its activation requires major conformational changes resulting in the exposure of its active site. Here, force-probe molecular dynamics simulations were used to obtain insight into the tension-induced activation mechanism. We find evidence for a sequential mechanically induced opening of the catalytic site without complete domain unfolding. Our results suggest the rupture of two terminal beta-sheets as the primary unfolding steps. The low force resistance of the C-terminal relative to the N-terminal beta-sheet is attributed to their different geometry. A subsequent rearrangement of the autoinhibitory tail is seen to lead to the exposure of the active site, as is required for titin kinase activity. These results support the hypothesis of titin kinase as a force sensor.

FIGURE 1

Physiological role, structure, and simulation setup of titin kinase. (a) Half of the sarcomere and the position of titin kinase (red) are sketched. Tandem titin domains are shown as spheres. (b) Titin kinase (1TKI) (Mayans et al., 1998) in its autoinhibited conformation. The catalytic site is shown in blue, the regulatory tail in red, and the terminal β-sheets in yellow. The harmonic pulling potentials used for the force-probe simulations described in the Methods section are symbolized as springs. (c) Simulation system of the FPMD simulations. All proteins were plotted with Pymol (DeLano, 2001).

FIGURE 2

Sequence alignment of the N-terminal regions of titin kinase and twitchin kinase. Invariant and semiinvariant residues are highlighted with a dark shaded and light shaded background, respectively. The numbering refers to twitchin kinase.

FIGURE 3

(a–c) Snapshots of titin kinase at 3.7 ns and 6.0 ns, and of twitchin kinase at 2.7ns. (d) Comparison of the side-chain fluctuations of the titin kinase (main panel) and twitchin kinase catalytic sites (inset). Center-of-mass distances between the side chains of Asp¹²⁷ and Arg¹²⁹ (black) and of Arg¹²⁹ and Glu¹³¹ (red) of titin kinase are shown. The inset shows the respective distances between Asp¹⁵² and Lys¹⁵⁴ (black) and Lys¹⁵⁴ and Glu¹⁵⁶ (red) of twitchin kinase.

FIGURE 4

(a) Maximal rupture forces as a function of pulling velocity v for the N-terminus (black) and C-terminus (red). Error bars indicate the uncertainty in the maximal rupture force due to the choice of a Gaussian distribution width for smoothing (see Methods section). (b) Spring positions (dislocation with respect to the starting position) at which the first (solid) and final (dashed) rupture of two N-terminal (black) and two C-terminal (gray) β-sheets occur, as a function of the pulling velocity for the four slowest pulling simulations. The inset shows the maximal rupture force observed during the period of the four β-sheet ruptures.

FIGURE 5

Force profiles and main rupture events for FPMD simulations of the low velocity regime. N-terminal ruptures (I) are shown in yellow and red, C-terminal ruptures (II) in light and dark cyan, and the αR2 rupture (III) in gray.

FIGURE 6

Open-closure motion of the small and big lobe during pulling. (a) Projection onto the second eigenvector as a function of simulation time. (b) Overlay of the extreme projections of the titin kinase motion on the second eigenvector (gray, closed conformation; colored, open conformation). The hinge axis between the two dynamic domains as determined with DynDom (Hayward and Berendsen, 1998) (cyan, big lobe; yellow and orange, small lobe and regulatory tail) is depicted as red arrow; hinge residues are shown in red.

FIGURE 7

Hydrogen bond interactions at the terminal β-sheets. Shown are hydrogen bond strengths for each hydrogen bond between the β-strands as indicated. As can be seen, βC1-βC2 ruptures simultaneously, the other sheets sequentially. For simultaneous rupture, subsequent transient hydrogen bonds are seen.

FIGURE 8

Differences of hydrogen bond rupture patterns for the two terminal β-sheets. (Upper panel) Sketch of the sequence of ruptures at the N-terminus (top) and the C-terminus (bottom). (Lower panel) Snapshots of the N-terminal βC1 and βC2 (yellow and green, respectively, top); snapshots of the C-terminal βC10 and βR1 (yellow and green, respectively, bottom). Hydrogen bonds are drawn as dashed (intact) and dotted (ruptures briefly before) dashed lines.

FIGURE 9

Force-probe Monte Carlo simulations with model energy landscapes. (a) Sketch of the model system: two attached β-sheets rupturing concurrently (top) and sequentially (bottom). (b) Assumed one-dimensional energy profiles for the sequential and concurrent rupture of a β-sheet with six hydrogen bonds. (c) Combined hydrogen bond potential energy surface G(x₁, x₂ − x₁). Here, x₁ is the reaction coordinate of the sequential, x₂ − x₁ that of the concurrent rupture, respectively, combining the profiles of b. (d) Spring positions at which the first (lower curve) and final rupture (upper curve) of the β-sheets occur as a function of the pulling velocity for the concurrent (○) and the sequential (+) rupture. The inset shows data for a larger velocity range.

FIGURE 10

Difference between fast and slow hydrogen bond ruptures. (a) Hydrogen bond potentials (black), running average over 50 ps and fit to the running average with a logistic function (shaded, see Methods section). Shown are two representative hydrogen bond ruptures at the N- and C-termini, respectively. (b) Width a₀ of the fitting function as a measure for the abruptness of the rupture process. Values are averaged over the hydrogen bonds of the sheet; error bars give the variance among them.

FIGURE 11

One-step rupture of the inhibitory tail from the small lobe. The van der Waals interaction energy for residue pairs of the small lobe (1–83) and αR2 (296–301), smoothed by Gaussian filtering with a band width of 100 ps, is shown.

FIGURE 12

Integrity of the active site. (a) C_α-distances of ATP binding residues. Straight lines are the equivalent distances in twitchin kinase (Kobe et al., 1996) and extracellular regulated kinase (ERK-2) (Zhang et al., 1994). (b) Displacement of the Gly-rich loop shown as root-mean square deviation of residues 14–19 from the starting structure. (c and d) Snapshots of the active site before (0 ns) and after (22 ns) the release of autoinhibition. ATP binding and catalytic residues are shown as sticks, the regulatory tail in red, and the Gly-rich loop in blue.

FIGURE 13

(Upper panel) Force profile as obtained from the twitchin kinase pulling simulation with 0.008 Å/ps velocity after Gaussian filtering. (Lower panel) Comparison of the terminal β-sheet dislocation between twitchin kinase and titin kinase during the first 6 ns of the 0.008 Å/ps pulling simulation.

FIGURE 14

Comparison of the pulling geometry between twitchin kinase and titin kinase. (a and b) Snapshots of the twitchin kinase pulling simulation at 0 ns and 6 ns. (c) Titin kinase starting structure. In a–c, the big lobe is shown in cyan, the small lobe in yellow, and the pulled C_α-atoms as spheres in red. (d) Angle between the force vector during the twitchin kinase pulling simulation and the initial force vector for titin kinase pulling simulation.