Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase

Abstract

Titin-like kinases are an important class of cytoskeletal kinases that intervene in the response of muscle to mechanical stimulation, being central to myofibril homeostasis and development. These kinases exist in autoinhibited states and, allegedly, become activated during muscle activity by the elastic unfolding of a C-terminal regulatory segment (CRD). However, this mechano-activation model remains controversial. Here we explore the structural, catalytic, and tensile properties of the multidomain kinase region of Caenorhabditis elegans twitchin (Fn(31)-Nlinker-kinase-CRD-Ig(26)) using X-ray crystallography, small angle X-ray scattering, molecular dynamics simulations, and catalytic assays. This work uncovers the existence of an inhibitory segment that flanks the kinase N-terminally (N-linker) and that acts synergistically with the canonical CRD tail to silence catalysis. The N-linker region has high mechanical lability and acts as the primary stretch-sensor in twitchin kinase, while the CRD is poorly responsive to pulling forces. This poor response suggests that the CRD is not a generic mechanosensor in this kinase family. Instead, the CRD is shown here to be permissive to catalysis and might protect the kinase active site against mechanical damage. Thus, we put forward a regulatory model where kinase inhibition results from the combined action of both N- and C-terminal tails, but only the N-terminal extension undergoes mechanical removal, thereby affording partial activation. Further, we compare invertebrate and vertebrate titin-like kinases and identify variations in the regulatory segments that suggest a mechanical speciation of these kinase classes.

Fig. 1.

Crystal structure of TwcKR (A) Domain composition of twitchin’s C terminus. Domains studied in this work are colored; (B) Crystal structure in three rotating views. Helix αR2 that blocks the ATP binding site is indicated with a white pointer.

Fig. 2.

The NL flanking region. (A) Hydrophobic groups mediating the packing of the NL against the kinase domain are listed. The buried salt bridge fixing the NL front to the kinase is highlighted; (B) Structural features of the NL segment in two views. Residues shown (or proposed to) undergo phosphorylation are in green. The strictly conserved tyrosine Y129 and its interacting residues are displayed. The cluster of positively charged residues (104–108) is shown. The catalytic glutamate in helix H3 is in magenta; (C) Motif conservation in the NL region. Sequences belong to TwcKs from C. elegans (Q23551), Mytilus galloprovincialis (Q7YT99), and Aplysia californica (Q16980); projectin from Drosophila melanogaster (O76281) and crayfish (Q86GD6); human (Q8WZ42) and mouse (A2ASS6) titin; and TTN-1 from C. elegans (A7DT28). Conservation is highlighted in yellow and interacting residues joined by lines. Positively charged residues at the NL front and negatively charged residues in the βC4-βC5 loop of the N-terminal kinase lobe are blue and red, respectively. Tyrosine residues in this region (or the structurally complementary region in mollusks) are marked in green. The similarity of twitchins and projectins, but not titins or TTN-1, is noticeable. A certain co-variation of the β-hairpin and the βC4-βC5 loop is detected. (Full sequence alignment of twitchins and projectins in Fig. S3).

Fig. 3.

SAXS analysis. (A) Molecular parameters calculated from SAXS data. MM, R_g, D_max denote the molecular mass, radius of gyration, and maximum size, respectively. Parameters without superscripts are experimental values; superscripts AB, RB, and XT refer to ab initio and rigid-body fitted models (shown in D) and the crystal structure, respectively. MMcalc is the theoretical MM computed from the protein sequence. χ is the discrepancy between experimental data and those computed from models; (B) SAXS data are displayed as dots with error bars (grey), while curves computed from the crystallographic (blue) and the RB model shown in (D) (red) are given as solid lines; (C) Distance distribution function; (D) Ab initio (white spheres) and RB (red) models are shown superimposed on the crystal structure (blue). In this RB model, only domain Fn³¹ was permitted to vary its position, while the rest of the molecule was fixed; (E) R_g distributions for models from a random pool of 10⁵ structures (solid line) and optimized by EOM (dashed line).

Fig. 4.

TwcKR catalytic activity. (A) Domain boundaries of segments tested; (B) Diagram of catalytic traces and (C) relative enzymatic activities calculated by linear regression using points in the linear range (10–30 min; linear fit R² > 90%). Full-length TwcKR and the NL-kinase-CRD construct were similarly inactive and their activity unquantifiable. Thus, they are not explicitly plotted here.

Fig. 5.

MD simulations of TwcKR response to stretch. (A) Force extension curves and (B) stretch-induced conformational states corresponding to main mechanical events in simulations of TwcKR. The force peaks from primary unfolding events are labeled.