Twitchin Kinase
Fig 1: Structure of a portion of twitchin kinase, containing fibronectin type III, kinase and immunoglobulin domains, from Caenorhabditis elegans.
Muscles of all animals contain giant proteins. These proteins contain a kinase region that, in their resting state, cannot dock ATP due to the presence of an inhibitory alpha helix. In humans these proteins are called titin kinase; in nematode worms Caenorhabditis elegans these proteins are called twitchin kinase.
For titin or twitchin to function ATP needs to dock into their respective catalytic sites. As these proteins are located in muscle cells they experience mechanical tensions. It was thus postulated that their autoinhibition is likely lifted through the application of a mechanical force that come about from muscle tension. It was subsequently shown that this in indeed the case for the kinase domain of titin, but not for twitchin. The solution of the crystal structure of a 3-domain section of twitchin, which included the kinase region, by our collaborator Olga Mayans (U. Liverpool) presented us with an opportunity to investigate the activation of twitchin kinase.
Using the new crystal structure we were able to simulate the pulling forces that could be experienced by twitchin to investigate the activation of its kinase domain in the presence of the surrounding domains.
Structure of twitchin kinase
Fig 2: Twitchin kinase is inhibited by an alpha helix bound within
and by an FnIII domain bound across its catalytic cleft.The crystal structure of twitchin kinase contains three domains: a fibronectin III (FnIII) domaim, a kinase domain (Kin) and an immunoglobulin (Ig) domain. The new structure showed that the kinase domain is not only inhibited by a c-terminal alpha helix but also by the FnIII domain which is bound across the catalytic cleft (see Fig. 2). The similarity of twitchin kinase to titin kinase had led researchers to assume that their activation mechanisms are also similar. The new information highlighting the structural differences of twitchin and titin indicates that twitchin activation may require a stronger stretching force than titin.
Investigating the force response of twitchin kinase
By employing steered molecular dynamics (SMD) one can computationally determine whether twitchin can be activated by a stretching force or whether an additional mechanism is used. In our computational experiment we fixed the N-terminal end of the three-domain twitchin fragment and pulled with a constant velocity on the C-terminal end (for details see the publication listed below). Fig. 3 shows the initial and final states of the simulation.
Fig 3: The initial and final states of the SMD simulation extending twitchin
kinase.Simulation results
Fig 4: Results of the pulling simulation. A Force extension curve.
B Rupture points associated with peaks in the force extension curve.
kinase.The SMD simulation yields a force-extension curve that provides us with peak forces encountered while pulling on the protein. In the case of twitchin kinase we need to know what force is required to remove both the N-terminal FnIII domain and the C-terminal inhibitory alpha helix. By correlating the force-extension curve with the simulation trajectory we can obtain precisely the information that we need.
It turns out that the N-terminal FnIII domain is easily removed but the C-terminal inhibitory helix is not! Figure 4 shows the transitions that the structure undergoes during force extension. Before the C-terminal inhibitory helix can be completely removed the N-terminal lobe of the kinase domain unfolds completely, thus destroying the ATP binding site. This is due to a strong Arg-Asp salt bridge between the autoinhibitory helix and the C-terminal lobe of the kinase domain. After careful analysis we showed that at reasonable forces the FnIII domain is separated from the catalytic cleft and the autoinhibitory helix is in an extended state that would allow a cofactor to bind (a state between a4 and a5 shown in Fig. 4).
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