Calcium increases titin N2A binding to F-actin and regulated thin filaments

Dutta, Samrat; Tsiros, Christopher; Sundar, Sai Lavanyaa; Athar, Humra; Moore, Jeffrey R.; Nelson, Brent; Gage, Matthew J.; Nishikawa, Kiisa C.

doi:10.1038/s41598-018-32952-8

Cited by 87 publications

(90 citation statements)

References 51 publications

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“…Calcium is an important regulator of muscle activation and the I91 domain in titin has been previously shown to be more stable in the presence of Ca 2+ . We hypothesized that Ca 2+ ‐enhanced stability might explain the Ca 2+ ‐enhanced binding observed between a construct of the N2A region and actin filaments . We repeated the equilibrium stability experiments in the presence of 50 μM CaCl 2 (pCa 4.3) to test this hypothesis.…”

Section: Resultsmentioning

confidence: 96%

“…In one proposed model, the N2A region of titin binds to actin upon an influx of calcium ions (~pCa 4.5) that occurs with muscle activation . This model is supported by a recent study demonstrating the binding of a recombinantly expressed N2A protein to actin and that this binding is enhanced in the presence of calcium …”

Section: Introductionmentioning

confidence: 84%

“…Upon influx of calcium in the active state, however, it has been established that p94 is activated and dissociated from titin . The N2A region has demonstrated enhanced binding to actin in the presence of calcium but the exact binding site remains unknown. The dissociation of p94 may open up a new binding site for actin during in eccentric contractions.…”

Section: Discussionmentioning

confidence: 99%

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Differences in stability and calcium sensitivity of the Ig domains in titin's N2A region

et al. 2020

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Titin is a large filamentous protein that spans half a sarcomere, from Z-disk to M-line. The N2A region within the titin molecule exists between the proximal immunoglobulin (Ig) region and the PEVK region and protein-protein interactions involving this region are required for normal muscle function. The N2A region consists of four Ig domains (I80-I83) with a 105 amino acid linker region between I80 and I81 that has a helical nature. Using chemical stability measurements, we show that predicted differences between the adjacent Ig domains (I81-I83) correlate with experimentally determined differences in chemical stability and refolding kinetics. Our work further shows that I83 has the lowest ΔG unfolding , which is increased in the presence of calcium (pCa 4.3), indicating that Ca 2+ plays a role in stabilizing this immunoglobulin domain. The characteristics of N2A's three Ig domains provide insight into the stability of the binding sites for proteins that interact with the N2A region. This work also provides insights into how Ca 2+ might influence binding events involving N2A. K E Y W O R D Schemical stability, immunoglobulin domain, N2A, titin

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Section: Resultsmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

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Differences in stability and calcium sensitivity of the Ig domains in titin's N2A region

et al. 2020

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“…; Dutta et al . ). In future work, the definition of the structural basis of the tunability of titin stiffness in situ will be possible by combining sarcomere‐level mechanics and X‐ray diffraction in intact fibres from the frog muscle and, eventually, under the condition that the sarcomere‐level resolution is preserved, in demembranated fibres from mouse muscle with specific mutations in the relevant titin domains.…”

Section: Discussionmentioning

confidence: 97%

“…A way to test whether the undamped elasticity is due to the PEVK region could be to apply our protocols to investigate if the specific PKC-α-mediated changes in PEVK stiffness (Hidalgo et al 2009) imply similar changes in the undamped titin stiffness. Notably, other mechanisms beyond Ig domains could contribute to the structural dynamics that integrate our undamped stiffness measure and make titin a tunable spring, among them, either the presence of breakable intramolecular links that change titin contour length (Kellermayer et al 2001), or the dynamic binding of the PEVK region of titin to actin that would keep the length of the titin spring constant at different SLs by shifting the titin bonds along the actin filament Dutta et al 2018). In future work, the definition of the structural basis of the tunability of titin stiffness in situ will be possible by combining sarcomere-level mechanics and X-ray diffraction in intact fibres from the frog muscle and, eventually, under the condition that the sarcomere-level resolution is preserved, in demembranated fibres from mouse muscle with specific mutations in the relevant titin domains.…”

Section: Molecular Basis Of the I-band Spring Tunabilitymentioning

confidence: 99%

Contracting striated muscle has a dynamic I‐band spring with an undamped stiffness 100 times larger than the passive stiffness

Powers

Bianco

Pertici

et al. 2020

The Journal of Physiology

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Key points Fast sarcomere‐level mechanics in contracting intact fibres from frog skeletal muscle reveal an I‐band spring with an undamped stiffness 100 times larger than the known static stiffness. This undamped stiffness remains constant in the range of sarcomere length 2.7–3.1 µm, showing the ability of the I‐band spring to adapt its length to the width of the I‐band. The stiffness and tunability of the I‐band spring implicate titin as a force contributor that, during contraction, allows weaker half‐sarcomeres to equilibrate with in‐series stronger half‐sarcomeres, preventing the development of sarcomere length inhomogeneity. This work opens new possibilities for the detailed in situ description of the structural–functional basis of muscle dysfunctions related to mutations or site‐directed mutagenesis in titin that alter the I‐band stiffness. Abstract Force and shortening in the muscle sarcomere are due to myosin motors from thick filaments pulling nearby actin filaments toward the sarcomere centre. Thousands of serially linked sarcomeres in muscle make the shortening (and the shortening speed) macroscopic, while the intrinsic instability of in‐series force generators is likely prevented by the cytoskeletal protein titin that connects the thick filament with the sarcomere end, working as an I‐band spring that accounts for the rise of passive force with sarcomere length (SL). However, current estimates of titin stiffness, deduced from the passive force–SL relation and single molecule mechanics, are much smaller than what is required to avoid the development of large inhomogeneities among sarcomeres. In this work, using 4 kHz stiffness measurements on a population of sarcomeres selected along an intact fibre isolated from frog skeletal muscle contracting at different SLs (temperature 4°C), we measure the undamped stiffness of an I‐band spring that at SL > 2.7 µm attains a maximum constant value of ∼6 pN nm−1 per half‐thick filament, two orders of magnitude larger than expected from titin‐related passive force. We conclude that a titin‐like dynamic spring in the I‐band, made by an undamped elastic element in‐series with damped elastic elements, adapts its length to the SL with kinetics that provide force balancing among serially linked sarcomeres during contraction. In this way, the I‐band spring plays a fundamental role in preventing the development of SL inhomogeneity.

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Titin UN2A Acts as a Stable, Non‐Polymorphic Scaffold in its Binding to CARP

Stehle,

Fleming,

Bauer

et al. 2023

ChemBioChem

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The N2A segment of titin functions as a pivotal hub for signal transduction and interacts with various proteins involved in structural support, chaperone activities, and transcriptional regulation. Notably, the “unique N2A” (UN2A) subdomain has been shown to interact with the stress‐regulated cardiac ankyrin repeat protein (CARP), which contributes to the regulation of sarcomeric stiffness. Previously, the UN2A domain's three‐dimensional structure was modelled based on its secondary structure content identified by NMR spectroscopy, considering the domain in isolation. In this study, we report experimental long‐range distance distributions by electron paramagnetic resonance (EPR) spectroscopy between the three helixes within the UN2A domain linked to the immunoglobulin domain I81 in the presence and absence of CARP. The data confirm the central three‐helix bundle fold of UN2A and show that this adopts a compact and stable conformation in absence of CARP. After binding to CARP, no significant conformational change was observed, suggesting that the UN2A domain retains its structure upon binding to CARP thereby, mediating the interaction approximately as a rigid‐body.

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Calcium increases titin N2A binding to F-actin and regulated thin filaments

Cited by 87 publications

References 51 publications

Differences in stability and calcium sensitivity of the Ig domains in titin's N2A region

Differences in stability and calcium sensitivity of the Ig domains in titin's N2A region

Contracting striated muscle has a dynamic I‐band spring with an undamped stiffness 100 times larger than the passive stiffness

Titin UN2A Acts as a Stable, Non‐Polymorphic Scaffold in its Binding to CARP

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