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. molecular mechanobiology | phospho-transfer catalysis | steered molecular dynamics simulations M echanical signals generated during physical activity are critical to the development and regulation of muscle tissue, which undergoes constant adaptation to mechanical demand. Despite the physiological importance of this mechano-feedback, little is known about how mechanical signals are sensed by the myofibril and further translated into biochemical events that feed into the homeodynamics of the tissue. In this context, the giant proteins of the titin-like family (0.7-4 MDa) are emerging as key mechanotransducers in muscle. Proteins from this family include titin and obscurin in mammals; twitchin, the obscurin homolog UNC-89 and the small TTN-1 titin in nematodes and mollusks; projectin and stretchin in insects (1, 2). These proteins are composed of numerous Ig-like domains linked in series and form long filaments embedded in the sarcoskeleton. There, these proteins mediate passive mechanical processes that dictate myofibrillar elasticity and relaxation rates.Titin-like proteins contain a conserved kinase region near their C terminus that consists of Ig-Ig-Fn-linker-kinase-tail-Ig domains. This conservation suggests that titin-like kinases are important members of signaling networks in the sarcomere, although little is known about their cellular context. Titin kinase...

Development, structure, and maintenance of C. elegans body wall muscle

“…The crystal structure, in vitro kinase assays, and SMDs of a larger segment of C. elegans twitchin (Fn-NL-kinase-CRD-Ig), indicate that the regulation of twitchin kinase activity is more complex than previously appreciated (von Castelmur et al, 2012). The structure (Figure 9) revealed for the first time that an N-terminal linker (NL), of 45 residues that lies between the Fn and kinase catalytic core, forms a "crown" that rests on the back of the interlobular kinase hinge region, with its remaining chain folded against the N-terminal lobe, sprawling across the β1-β2 hairpin (containing the glycine-rich loop in the ATP binding pocket).…”

Binding partners of the kinase domains inDrosophilaobscurin and their effect on the structure of the flight muscle

“…The regulatory domain of Kin1, taken out of its usual context, might not associate with the active site in the same way as it would in vivo. Alternatively, the regulatory domain might be required to stabilise the kinase structure when the muscle is stretched, as suggested for twitchin kinase (von Castelmur et al, 2012).…”

“…Mechano-sensing by the kinase can result in changes in the C-terminal regulatory domain and transient binding of ligands to the kinase scaffold. The precise mechanism of regulation varies in different species (Lange et al, 2005;Mayans et al, 2013;Puchner et al, 2008;von Castelmur et al, 2012).…”

“…This sequence can inhibit an active kinase, or regulate ligand binding; it can also be part of the structure of the kinase domain, and necessary to maintain the stability of the domain (Gautel, 2011a;Mayans et al, 2013;von Castelmur et al, 2012). Titin-like kinases are linked to stretch-activated signalling pathways in muscle.…”

Time-Resolved Resonance Raman Spectroscopy and Application to Studies on Ultrafast Protein Dynamics

“…For example, mechano-sensitive enzymes, such as titin-like kinases, have been investigated. 158,159 The enzyme-DNA chimeras, in which a DNA molecular spring attached to the enzyme exerts a force in a known direction, were used for measurements of the kinetics of catalyzed reactions under a non-destructive mechanical stress on an enzyme. 160164 An asymmetric effect of mechanical stress on the forward and reverse reactions catalyzed by an enzyme was reported recently.…”