| Activation of the titin kinase, the catalytic domain of the muscle protein titin, requires major conformational rearrangements resulting in the exposure of its phosphorylation site. It can be assumed but has not yet been shown that the requisite structural change is caused by stretched titin passing down the tension to the titin kinase. Force probe molecular dynamics simulations can give a detailed description of the activation mechanism and, hence, can test the hypothesis that titin kinase is the force sensor for the muscle cell. |
| Titin, a filament of the muscle cell, is a giant protein of approx. 3000 kDa, spans half the sarcomere, and is the longest covalently linked protein known [1]. Titin is composed of approx. 300 repeating domains, which mainly are similar to immunoglobulin and fibronectin, and one enzymatic domain: the titin kinase (Fig. 1). | |
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| Fig. 1 A single titin molecule. One dot represents an immunoglobulin or fibronectin like domain (N2B and PEVK domains are omitted). | |
Fig. 2 Titin connects myosin and actin in the sarcomere. Click on the figure for an animated gif demonstrating titin unfolding upon sarcomere stretching.. | The titin filament unfolds upon muscle cell stretching and refolds upon relaxation, thereby realigning the sarcomere and giving the muscle cell its elasticity [1] (fig. 2). A crystal structure of the titin-kinase domain is solved and shown in Fig. 3 [2]. Activation of the titin kinase requires two steps: phosphorylation of tyrosine 170 and binding of calcium/calmodulin. The main feature of the activation mechanism is the need of large-scale structural rearrangement to expose the buried tyrosine to solvent prior to or during its phosphorylation. |
Fig. 3. Ribbon diagram of the structure of titin kinase, showing the autoinhibited form with the regulatory tail (red) blocking the active site and Tyr170 (green) located at the P+1 loop (yellow). |
Located near the C-terminus the titin kinase is under the influence of tension of unfolded titin. An obvious assumption is that the active force of the filamentous titin molecule triggers this major conformational change in the titin kinase. In other words, in response to a force of certain magnitude, partly unfolded titin domains could pass down the mechanical stress to the titin kinase. The subsequent structural rearrangement could relieve the autoinhibition followed by tyrosine phosphorylation. |
| We are aiming at understanding the molecular mechanism and energetics of the titin kinase activation induced by the pulling force of a titin molecule in a stretched muscle cell. Force probe molecular dynamics will be used to complement the experimental results of atomic force microscopy on titin kinase (for AFM of other titin domains see [3]). | |
| Fig.4 Force probe molecular dynamics: Subjecting the N and C-termini to a harmonic spring potential modelling the tension of a stretched titin filament. |
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References:
[1] Furst, DO, Osborn, M, Nave, R, Weber, K (1988). The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z line extends close to the M line. J. Cell. Biology 106, 1563-1572.. [2] Mayans, O, van der Ven, PF, Wilm, M, Mues, A, Young, P, Furst, DO et al.. (1998). Structural basis for activation of the titin kinase domain during myofibrillogenesis. Nature 395, 863-869. [3] Rief, M, Gautel, M, Oesterhelt, F, Fernandez, JM, Gaub, HE (1997). Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109-1112. |