Titin Domains Research Articles

Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.

Structural basis of domain destabilization by dilated-cardiomyopathy-causing missense mutations in titin.

Truncating variants in the titin gene are the most common cause of familial dilated cardiomyopathy (DCM), accounting for 25% of the genotype-positive cases. Similar to other genetic diseases triggered by gene loss of function, it has been proposed that destabilizing titin missense mutations can also result in DCM. Indeed, the highly destabilizing C3575S mutation targeting immunoglobulin-like (Ig) 21 domain of cardiac titin has been recently identified as a cause of DCM. However, the effects of the mutation on the structure of the parent domain and whether altered disulfide bond formation contribute to the destabilizing properties of the mutation remain unexplored. Here, using high-resolution X-ray crystallography, we report that the structure of the C3575S mutant domain is largely unperturbed (RMSD: 0.538 Å). In addition, using gel-shift assays, we have ruled out that the lower thermal stability of the mutant domain results from its inability to establish intramolecular disulfide bonds. Instead, we propose that the lower hydrophobicity of serine is not well accommodated in the core of mutant domains, causing their destabilization. Finally, we have observed that similar missense mutations, which cause a decrease in hydrophobicity in buried positions of titin Ig domains, are enriched in DCM patients. Altogether, our results illustrate that subtle physicochemical changes induced by titin missense mutations can cause domain destabilization, even in the absence of remarkable structural alterations, resulting in development of DCM.

Stability of tandem Ig domain unfolding within proximal Ig segment of titin.

Titin is a giant filamentous protein contributing to the passive elasticity of muscle and is composed of approximately 300 serially linked immunoglobulin-like domains. The tandem domains of titin's proximal immunoglobulin (Ig) segment have been shown to separate at relatively low forces and thus contribute to the protein's elasticity. While titin's elastic behavior is attributed to extension of the proximal Ig segment and PEVK region beyond passive slack length, the potential unfolding of individual Ig domains has been posited to contribute to even greater extensibility. Computational modeling and mechanical stability testing have demonstrated Ig domain unfolding under physiologically relevant force, with neighboring domains exhibiting variable degrees of stability. Thermal and chemical stability experiments were conducted on domains I65-I70 of the proximal tandem Ig segment to evaluate the stability of adjacent domains. Testing revealed variable free energies of unfolding, with I65 demonstrating the lowest thermal and chemical stability out of all domains of interest, and I67 demonstrating the highest. The quantification of each domain's relative stability helps elucidate the role of the proximal Ig segment in titin's elasticity and can be used to assess the unfolding potential of other domains along the protein's extensible regions.

Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations.

A highly bioluminescent protein, NanoLuc (Nluc), has seen numerous applications in biological assays since its creation. We recently engineered a NanoLuc polyprotein that showed high bioluminescence but displayed a strong misfolding propensity after mechanical unfolding. Here, we present our single-molecule force spectroscopy (SMFS) studies by atomic force microscopy (AFM) and steered molecular dynamics (SMD) simulations on two new hybrid protein constructs comprised of Nluc and I91 titin domains, I91-I91-Nluc-I91-I91-I91-I91 (I912-Nluc-I914) and I91-Nluc-I91-Nluc-I91-Nluc-I91, to characterize the unfolding behavior of Nluc in detail and to further investigate its misfolding properties that we observed earlier for the I912-Nluc3-I912 construct. Our SMFS results confirm that Nluc's unfolding proceeds similarly in all constructs; however, Nluc's refolding differs in these constructs, and its misfolding is minimized when Nluc is monomeric or separated by I91 domains. Our simulations on monomeric Nluc, Nluc dyads, and Nluc triads pinpointed the origin of its mechanical stability and captured interesting unfolding intermediates, which we also observed experimentally.

Direct and Simultaneous Measurement of the Stiffness and Internal Friction of a Single Folded Protein.

The nanomechanical response of a folded single protein, the natural nanomachine responsible for myriad biological processes, provides insight into its function. The conformational flexibility of a folded state, characterized by its viscoelasticity, allows proteins to adopt different shapes to perform their function. Despite efforts, its direct measurement has not been possible so far. We present a direct and simultaneous measurement of the stiffness and internal friction of the folded domains of the protein titin using a special interferometer based atomic force microscope. We analyzed the data by carefully separating different contributions affecting the response of the experimental probe to obtain the folded state's viscoelasticity. Above ∼95 pN of force, the individual immunoglobulins of titin transition from an elastic solid-like native state to a soft viscoelastic intermediate.

Use of animal models to understand titin physiology and pathology.

In recent years, increasing attention has been paid to titin (TTN) and its mutations. Heterozygous TTN truncating variants (TTNtv) increase the risk of a cardiomyopathy. At the same time, TTNtv and few missense variants have been identified in patients with mainly recessive skeletal muscle diseases. The pathogenic mechanisms underlying titin‐related diseases are still partly unknown. Similarly, the titin mechanical and functional role in the muscle contraction are far from being exhaustively clarified. In the last few years, several animal models carrying variants in the titin gene have been developed and characterized to study the structural and mechanical properties of specific titin domains or to mimic patients' mutations. This review describes the main animal models so far characterized, including eight mice models and three fish models (Medaka and Zebrafish) and discusses the useful insights provided by a thorough characterization of the cell‐, tissue‐ and organism‐phenotypes in these models.

Interdomain Linker Effect on the Mechanical Stability of Ig Domains in Titin

Titin is the largest protein in humans, composed of more than one hundred immunoglobulin (Ig) domains, and plays a critical role in muscle’s passive elasticity. Thus, the molecular design of this giant polyprotein is responsible for its mechanical function. Interestingly, most of these Ig domains are connected directly with very few interdomain residues/linker, which suggests such a design is necessary for its mechanical stability. To understand this design, we chose six representative Ig domains in titin and added nine glycine residues (9G) as an artificial interdomain linker between these Ig domains. We measured their mechanical stabilities using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) and compared them to the natural sequence. The AFM results showed that the linker affected the mechanical stability of Ig domains. The linker mostly reduces its mechanical stability to a moderate extent, but the opposite situation can happen. Thus, this effect is very complex and may depend on each particular domain’s property.

GSK-3β Localizes to the Cardiac Z-Disc to Maintain Length Dependent Activation.

Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. Inducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

E3-ligase knock down revealed differential titin degradation by autophagy and the ubiquitin proteasome system

The sarcomere protein titin is a major determinant of cardiomyocyte stiffness and ventricular distensibility. The constant mechanical stress on titin requires well-controlled protein quality control, the exact mechanisms of which have not yet been fully elucidated. Here, we analyzed E3-ligases potentially responsible for cardiac titin ubiquitination and specifically studied the involvement of the autophagosomal system in titin degradation. Pharmacological inhibition of autophagy and the proteasome in cultured primary rat cardiomyocytes significantly elevated titin ubiquitination and increased titin degradation. Using in-vitro pull down assays we identified binding of E3-ligases MuRF1-3, CHIP and Fbx32 to several titin domains. Immunofluorescence analysis showed sarcomeric localization of the E3-ligases. siRNA-mediated knock-down of the E3-ligases MuRF-1, -3 and a combination of CHIP/Fbx32 significantly reduced autophagy-related titin ubiquitination, whereas knock-down of MuRF-2 and -3 reduced proteasome-related titin ubiquitination. We demonstrated that the proteasomal and the autophagosomal-lysosomal system participate in degradation of the titin filament. We found that ubiquitination and degradation of titin are partially regulated by E3-ligases of the MuRF family. We further identified CHIP and Fbx32 as E3-ligases involved in titin ubiquitination.

Protein Unfolding: Denaturant vs. Force.

While protein refolding has been studied for over 50 years since the pioneering work of Christian Anfinsen, there have been a limited number of studies correlating results between chemical, thermal, and mechanical unfolding. The limited knowledge of the relationship between these processes makes it challenging to compare results between studies if different refolding methods were applied. Our current work compares the energetic barriers and folding rates derived from chemical, thermal, and mechanical experiments using an immunoglobulin-like domain from the muscle protein titin as a model system. This domain, I83, has high solubility and low stability relative to other Ig domains in titin, though its stability can be modulated by calcium. Our experiments demonstrated that the free energy of refolding was equivalent with all three techniques, but the refolding rates exhibited differences, with mechanical refolding having slightly faster rates. This suggests that results from equilibrium-based measurements can be compared directly but care should be given comparing refolding kinetics derived from refolding experiments that used different unfolding methods.

OTHER NMDs: EP.360 Is7 domain of titin is essential for cardiac function in mice

OTHER NMDs: EP.360 Is7 domain of titin is essential for cardiac function in mice

Unexpected Low Mechanical Stability of Titin I27 Domain at Physiologically Relevant Temperature.

The extensively studied immunoglobulin (Ig) domain I27 of the giant force-bearing protein titin has provided a basis for our current understanding of the structural stability, dynamics, and function of the numerous mechanically stretched Ig domains in the force-bearing I-band of titin. The current consensus is that titin I27 has a high mechanical stability characterized by very low unfolding rate (<10-3 s-1) in physiological force range and high unfolding forces (>100 pN) at typical physiological force loading rates from experiments at typical laboratory temperatures. Here, we report that when the temperature is increased from 23 to 37 °C, the unfolding rate of I27 drastically increases by ∼100-fold at the physiological level of forces, indicating a low mechanical stability of I27 at physiological conditions. The result provides new insights into the structural states and the associated functions of I27 and other similar titin I-band Ig domains.

Why exercise builds muscles: titin mechanosensing controls skeletal muscle growth under load

Muscles sense internally generated and externally applied forces, responding to these in a coordinated hierarchical manner at different timescales. The center of the basic unit of the muscle, the sarcomeric M-band, is perfectly placed to sense the different types of load to which the muscle is subjected. In particular, the kinase domain of titin (TK) located at the M-band is a known candidate for mechanical signaling. Here, we develop a quantitative mathematical model that describes the kinetics of TK-based mechanosensitive signaling and predicts trophic changes in response to exercise and rehabilitation regimes. First, we build the kinetic model for TK conformational changes under force: opening, phosphorylation, signaling, and autoinhibition. We find that TK opens as a metastable mechanosensitive switch, which naturally produces a much greater signal after high-load resistance exercise than an equally energetically costly endurance effort. Next, for the model to be stable and give coherent predictions, in particular for the lag after the onset of an exercise regime, we have to account for the associated kinetics of phosphate (carried by ATP) and for the nonlinear dependence of protein synthesis rates on muscle fiber size. We suggest that the latter effect may occur via the steric inhibition of ribosome diffusion through the sieve-like myofilament lattice. The full model yields a steady-state solution (homeostasis) for muscle cross-sectional area and tension and, a quantitatively plausible hypertrophic response to training, as well as atrophy after an extended reduction in tension.

Crystallographic Structures of Titin Immunoglobulin-Like I21 Domains Involved in Dilated Cardiomyopathy

The protein titin is the main responsible for the passive elasticity of the sarcomere, determining the stiffness and contractile function of cardiomyocytes. Titin is also a key factor in the etiology of heart disease, given that truncating mutations in the titin gene are the most common cause of dilated cardiomyopathy (DCM). However, missense mutations have commonly been classified as variants of uncertain significance because of their high frequency in the general population and the absence of functional annotation. We have recently identified a missense mutation targeting a conserved cysteine in the I21 domain of titin as a cause of DCM in humans. Here, we present the structural characterization of the wild type domain, that follows a fold characteristic for the intermediate set (type I) of the immunoglobulin superfamily: 2 antiparallel 4-strand-β-sheets packed in a β-sandwich shape. Interestingly, we observed that the I21 titin domain contains a second cysteine located 5Å away from the cysteine position mutated in dilated cardiomyopathy patients. We also report the structural and biophysical characterization of the mutated domain. The mutated domain presents unaltered overall structure, although its thermal stability is severely compromised, showing a decrease of 17ºC in its melting temperature when compared to the wild type domain. Our results suggest that titin domain destabilization by missense variants can lead to DCM, even if the structure of the native state is preserved. Future research will investigate the origin of the thermal destabilization in the mutated domain, and the potential influence of intramolecular disulfide bonds.

Does ATP Modulate Protein Nanomechanics

ATP has a crucial role in cell biology. One of its well-known functions is intracellular energy transport to fuel many different cellular processes. However, another role for this molecule has been recently described. ATP has been proposed to be also a biological hydrotrope, preventing protein aggregation and dissolving already formed aggregates. The heart muscle is constantly contracting and relaxing for a lifetime, which means, that it is a highly energy-demanding organ. This contractility is carried out by the sarcomeres, functional units made of proteins with mechanical functions, such as titin. The immunoglobulin-like domains of this protein are able to unfold and refold under force during contraction. We hypothesized that the mechanical properties of these domains may be affected by the hydrotropic function of ATP. To test this hypothesis, we measured how I27 domain of titin unfolds and refolds in absence and presence of ATP using Atomic Force Spectroscopy (AFS). We subjected polyproteins composed of eight domains of I27 to unfolding-quench-probe experiments. These experiments inform both about the mechanical stability of I27 and its ability to refold. Contrary to our initial hypothesis, we have found that the presence of 10 mM ATP does not affect the mechanical stability or the folding rate of titin I27 domain.

When is an obscurin variant pathogenic? The impact of Arg4344Gln and Arg4444Trp variants on protein–protein interactions and protein stability

Obscurin is a giant muscle protein that connects the sarcomere with the sarcoplasmic reticulum, and has poorly understood structural and signalling functions. Increasingly, obscurin variants are implicated in the pathophysiology of cardiovascular diseases. The Arg4344Gln variant (R4344Q) in obscurin domain Ig58, initially discovered in a patient with hypertrophic cardiomyopathy, has been reported to reduce binding to titin domains Z8-Z9, impairing obscurin’s Z-disc localization. An R4344Q knock-in mouse developed a cardiomyopathy-like phenotype with abnormal Ca2+-handling and arrhythmias, which were attributed to an enhanced affinity of a putative interaction between obscurin Ig58 and phospholamban (PLN) due to the R4344Q variant. However, the R4344Q variant is found in 15% of African Americans, arguing against its pathogenicity. To resolve this apparent paradox, we quantified the influence of the R4344Q variant (alongside another potentially pathogenic variant: Arg4444Trp (R4444W)) on binding to titin Z8-Z9, novex-3 and PLN using pull-down assays and microscale thermophoresis and characterized the influence on domain stability using differential scanning fluorimetry. We found no changes in titin binding and thermostability for both variants and modestly increased affinities of PLN for R4344Q and R4444W. While we could not confirm the novex-3/obscurin interaction, the PLN/obscurin interaction relies on the transmembrane region of PLN and is not reproducible in mammalian cells, suggesting it is an in vitro artefact. Without clear clinical evidence for disease involvement, we advise against classifying these obscurin variants as pathogenic.

Calibration of T-shaped atomic force microscope cantilevers using the thermal noise method.

The tip-sample interaction force measurements in atomic force microscopy (AFM) provide information about materials' properties with nanoscale resolution. The T-shaped cantilevers used in Torsional-Harmonic AFM allow measuring the rapidly changing tip-sample interaction forces using the torsional (twisting) deflections of the cantilever due to the off-axis placement of the sharp tip. However, it has been difficult to calibrate these cantilevers using the commonly used thermal noise-based calibration method as the mechanical coupling between flexural and torsional deflections makes it challenging to determine the deflection sensitivities from force-distance curves. Here, we present thermal noise-based calibration of these T-shaped AFM cantilevers by simultaneously analyzing flexural and torsional thermal noise spectra, along with deflection signals during a force-distance curve measurement. The calibration steps remain identical to the conventional thermal noise method, but a computer performs additional calculations to account for mode coupling. We demonstrate the robustness of the calibration method by determining the sensitivity of calibration results to the laser spot position on the cantilever, to the orientation of the cantilever in the cantilever holder, and by repeated measurements. We validated the quantitative force measurements against the known unfolding force of a protein, the I91 domain of titin, which resulted in consistent unfolding force values among six independently calibrated cantilevers.

The Axial Alignment of Titin on the Muscle Thick Filament Supports Its Role as a Molecular Ruler

The giant protein titin is expressed in vertebrate striated muscle where it spans half a sarcomere from the Z-disc to the M-band and is essential for muscle organisation, activity and health. The C-terminal portion of titin is closely associated with the thick, myosin-containing filament and exhibits a complex pattern of immunoglobulin and fibronectin domains. This pattern reflects features of the filament organisation suggesting that it acts as a molecular ruler and template, but the exact axial disposition of the molecule has not been determined. Here, we present data that allow us to precisely locate titin domains axially along the thick filament from its tip to the edge of the bare zone. We find that the domains are regularly distributed along the filament at 4-nm intervals and we can determine the domains that associate with features of the filament, such as the 11 stripes of accessory proteins. We confirm that the nine stripes ascribed to myosin binding protein-C are not related to the titin sequence previously assumed; rather, they relate to positions approximately 18 domains further towards the C terminus along titin. This disposition also allows a subgroup of titin domains comprising two or three fibronectin domains to associate with each of the 49 levels of myosin heads in each half filament. The results strongly support the role of titin as a blueprint for the thick filament and the arrangement of the myosin motor domains.

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.

Exploiting a Mechanical Perturbation of a Titin Domain to Identify How Force Field Parameterization Affects Protein Refolding Pathways.

Molecular mechanics force fields have been shown to differ in their predictions of biomolecular processes such as protein folding. To test how force field differences affect predicted polypeptide behavior, we created a mechanically perturbed model of the β-stranded I91 titin domain based on atomic force spectroscopy data and examined its refolding behavior using six different force fields. We found that different force fields varied significantly in their ability to refold the mechanically perturbed I91 domain. Examination of the perturbed I91 unfolded state revealed that all five Amber force fields oversample a specific region of the Ramachandran plot, thereby creating unfolded state intermediates which are not predicted by the Charmm 22* force field. Simulations of perturbed I91 refolding with Amber FB15 revealed that Amber FB15 destabilizes stable portions of I91, thereby contradicting experimental stability analyses. Finally, inspection of the perturbed I91 unfolded state along with equilibration simulations of the Ac-(AAQAA)3-NH2 peptide suggest that high dihedral torsional barriers cause the Amber ff14SB force field to predict higher helical lifetimes relative to other force fields. These results suggest that using mechanically perturbed models can provide a controlled method to gain insights into how force fields affect polypeptide behavior.