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The N2A region of titin has a unique structural configuration.

ABSTRACT: The N2A segment of titin is a main signaling hub in the sarcomeric I-band that recruits various signaling factors and processing enzymes. It has also been proposed to play a role in force production through its Ca2+-regulated association with actin. However, the molecular basis by which N2A performs these functions selectively within the repetitive and extensive titin chain remains poorly understood. Here, we analyze the structure of N2A components and their association with F-actin. Specifically, we characterized the structure of its Ig domains by elucidating the atomic structure of the I81-I83 tandem using x-ray crystallography and computing a homology model for I80. Structural data revealed these domains to present heterogeneous and divergent Ig folds, where I81 and I83 have unique loop structures. Notably, the I81-I83 tandem has a distinct rotational chain arrangement that confers it a unique multi-domain topography. However, we could not identify specific Ca2+-binding sites in these Ig domains, nor evidence of the association of titin N2A components with F-actin in transfected C2C12 myoblasts or C2C12-derived myotubes. In addition, F-actin cosedimentation assays failed to reveal binding to N2A. We conclude that N2A has a unique architecture that predictably supports its selective recruitment of binding partners in signaling, but that its mechanical role through interaction with F-actin awaits validation.

SUBMITTER: Stronczek C

PROVIDER: S-EPMC8042602 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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The N2A region of titin has a unique structural configuration.

Stronczek Chiara C Lange Stephan S Bullard Belinda B Wolniak Sebastian S Börgeson Emma E Mayans Olga O Fleming Jennifer R JR

The Journal of general physiology 20210701 7

The N2A segment of titin is a main signaling hub in the sarcomeric I-band that recruits various signaling factors and processing enzymes. It has also been proposed to play a role in force production through its Ca2+-regulated association with actin. However, the molecular basis by which N2A performs these functions selectively within the repetitive and extensive titin chain remains poorly understood. Here, we analyze the structure of N2A components and their association with F-actin. Specificall ...[more]

PMID: 33836065

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Project description:Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (Funfold). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se. Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling.

Project description:BACKGROUND:Titin is a giant elastic protein that spans the half-sarcomere from Z-disk to M-band. It acts as a molecular spring and mechanosensor and has been linked to striated muscle disease. The pathways that govern titin-dependent cardiac growth and contribute to disease are diverse and difficult to dissect. METHODS:To study titin deficiency versus dysfunction, the authors generated and compared striated muscle specific knockouts (KOs) with progressive postnatal loss of the complete titin protein by removing exon 2 (E2-KO) or an M-band truncation that eliminates proper sarcomeric integration, but retains all other functional domains (M-band exon 1/2 [M1/2]-KO). The authors evaluated cardiac function, cardiomyocyte mechanics, and the molecular basis of the phenotype. RESULTS:Skeletal muscle atrophy with reduced strength, severe sarcomere disassembly, and lethality from 2 weeks of age were shared between the models. Cardiac phenotypes differed considerably: loss of titin leads to dilated cardiomyopathy with combined systolic and diastolic dysfunction-the absence of M-band titin to cardiac atrophy and preserved function. The elastic properties of M1/2-KO cardiomyocytes are maintained, while passive stiffness is reduced in the E2-KO. In both KOs, we find an increased stress response and increased expression of proteins linked to titin-based mechanotransduction (CryAB, ANKRD1, muscle LIM protein, FHLs, p42, Camk2d, p62, and Nbr1). Among them, FHL2 and the M-band signaling proteins p62 and Nbr1 are exclusively upregulated in the E2-KO, suggesting a role in the differential pathology of titin truncation versus deficiency of the full-length protein. The differential stress response is consistent with truncated titin contributing to the mechanical properties in M1/2-KOs, while low titin levels in E2-KOs lead to reduced titin-based stiffness and increased strain on the remaining titin molecules. CONCLUSIONS:Progressive depletion of titin leads to sarcomere disassembly and atrophy in striated muscle. In the complete knockout, remaining titin molecules experience increased strain, resulting in mechanically induced trophic signaling and eventually dilated cardiomyopathy. The truncated titin in M1/2-KO helps maintain the passive properties and thus reduces mechanically induced signaling. Together, these findings contribute to the molecular understanding of why titin mutations differentially affect cardiac growth and have implications for genotype-phenotype relations that support a personalized medicine approach to the diverse titinopathies.

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The N2A region of titin has a unique structural configuration.

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The N2A region of titin has a unique structural configuration.

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