title: Mechanoenzymatics of titin kinase.
abstract: Biological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM-based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation and substrate turnover.
authors.affiliation: Chair for Applied Physics, Center for Integrated Protein Science Munich and Center for Nanoscience, Ludwig-Maximilians-Universität München, 80799 Munich, Germany.
authors.forename: Elias M
authors.initials: EM
authors.lastname: Puchner
authors.forename: Alexander
authors.initials: A
authors.lastname: Alexandrovich
authors.forename: Ay Lin
authors.initials: AL
authors.lastname: Kho
authors.forename: Ulf
authors.initials: U
authors.lastname: Hensen
authors.forename: Lars V
authors.initials: LV
authors.lastname: Schäfer
authors.forename: Birgit
authors.initials: B
authors.lastname: Brandmeier
authors.forename: Frauke
authors.initials: F
authors.lastname: Gräter
authors.forename: Helmut
authors.initials: H
authors.lastname: Grubmüller
authors.forename: Hermann E
authors.initials: HE
authors.lastname: Gaub
authors.forename: Mathias
authors.initials: M
authors.lastname: Gautel