Dietmar Labeit

Calcium-dependent molecular spring elements in the giant protein titin

Titin (also known as connectin) is a giant protein with a wide range of cellular functions, including providing muscle cells with elasticity. Its physiological extension is largely derived from the PEVK segment, rich in proline (P), glutamate (E), valine (V), and lysine (K) residues. We studied recombinant PEVK molecules containing the two conserved elements: ≈28-residue PEVK repeats and E-rich motifs. Single molecule experiments revealed that calcium-induced conformational changes reduce the bending rigidity of the PEVK fragments, and site-directed mutagenesis identified four glutamate residues in the E-rich motif that was studied (exon 129), as critical for this process. Experiments with muscle fibers showed that titin-based tension is calcium responsive. We propose that the PEVK segment contains E-rich motifs that render titin a calcium-dependent molecular spring that adapts to the physiological state of the cell.

Nebulin regulates thin filament length, contractility, and Z-disk structure in vivo

The precise assembly of the highly organized filament systems found in muscle is critically important for its function. It has been hypothesized that nebulin, a giant filamentous protein extending along the entire length of the thin filament, provides a blueprint for muscle thin filament assembly. To test this hypothesis, we generated a KO mouse model to investigate nebulin functions in vivo. Nebulin KO mice assemble thin filaments of reduced lengths and ∼15% of their Z‐disks are abnormally wide. Our data demonstrate that nebulin functions in vivo as a molecular ruler by specifying pointed‐ and barbed‐end thin filament capping. Consistent with the shorter thin filament length of nebulin deficient mice, maximal active tension was significantly reduced in KO animals. Phenotypically, the murine model recapitulates human nemaline myopathy (NM), that is, the formation of nemaline rods combined with severe skeletal muscle weakness. The myopathic changes in the nebulin KO model include depressed contractility, loss of myopalladin from the Z‐disk, and dysregulation of genes involved in calcium homeostasis and glycogen metabolism; features potentially relevant for understanding human NM.

Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2

The muscle‐specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4–18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild‐type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.

Multiple Molecular Interactions Implicate the Connectin/Titin N2A Region as a Modulating Scaffold for p94/Calpain 3 Activity in Skeletal Muscle

p94/calpain 3 is a skeletal muscle-specific Ca2+-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.

Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin

Titin forms an intrasarcomeric filament system in vertebrate striated muscle, which has elastic and signaling properties and is thereby central to mechanotransduction. Near its C‐terminus and directly preceding a kinase domain, titin contains a conserved pattern of Ig and FnIII modules (IgA168‐IgA169‐FnIIIA170, hereby A168‐A170) that recruits the E3 ubiquitin‐ligase MuRF‐1 to the filament. This interaction is thought to regulate myofibril turnover and the trophic state of muscle. We have elucidated the crystal structure of A168‐A170, characterized MuRF‐1 variants by circular dichroism (CD) and SEC‐MALS, and studied the interaction of both components by isothermal calorimetry, SPOTS blots, and pull‐down assays. This has led to the identification of the molecular determinants of the binding. A168‐A170 shows an extended, rigid architecture, which is characterized by a shallow surface groove that spans its full length and a distinct loop protrusion in its middle point. In MuRF‐1, a C‐terminal helical domain is sufficient to bind A168‐A170 with high affinity. This helical region predictably docks into the surface groove of A168‐A170. Furthermore, pull‐down assays demonstrate that the loop protrusion in A168‐A170 is a key mediator of MuRF‐1 recognition. Our findings indicate that this region of titin could serve as a target to attempt therapeutic inhibition of MuRF‐1‐mediated muscle turnover, where binding of small molecules to its distinctive structural features could block MuRF‐1 access.—Mrosek, M., Labeit, D., Witt, S., Heerklotz, H., von Castelmur, E., Labeit, S., Mayans, O. Molecular determinants for the recruitment of the ubiquitin‐ligase MuRF‐1 onto M‐line titin FASEB J. 21, 1383–1392 (2007)

A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain

Myofibril elasticity, critical to muscle function, is dictated by the intrasarcomeric filament titin, which acts as a molecular spring. To date, the molecular events underlying the mechanics of the folded titin chain remain largely unknown. We have elucidated the crystal structure of the 6-Ig fragment I65–I70 from the elastic I-band fraction of titin and validated its conformation in solution using small angle x-ray scattering. The long-range properties of the chain have been visualized by electron microscopy on a 19-Ig fragment and modeled for the full skeletal tandem. Results show that conserved Ig–Ig transition motifs generate high-order in the structure of the filament, where conformationally stiff segments interspersed with pliant hinges form a regular pattern of dynamic super-motifs leading to segmental flexibility in the chain. Pliant hinges support molecular shape rearrangements that dominate chain behavior at moderate stretch, whereas stiffer segments predictably oppose high stretch forces upon full chain extension. There, librational entropy can be expected to act as an energy barrier to prevent Ig unfolding while, instead, triggering the unraveling of flanking springs formed by proline, glutamate, valine, and lysine (PEVK) sequences. We propose a mechanistic model based on freely jointed rigid segments that rationalizes the response to stretch of titin Ig-tandems according to molecular features.

Suppressed Disassembly of Autolyzing p94/CAPN3 by N2A Connectin/Titin in a Genetic Reporter System

p94/calpain 3 is a skeletal muscle-specific member of the Ca2+-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent β-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.

Mechanical Properties of Titin Isoforms

Titin is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the sarcomere. As a result of differential splicing, length variants of titin are expressed in different skeletal and cardiac muscles. Here we first briefly review some of our previous work that has revealed that titin develops force in sarcomeres either stretched beyond their slack length (passive force) or shortened to below the slack length (restoring force) and that titins force underlies a large fraction of the diastolic force of cardiac muscle. Next we present our mechanical and immunoelectron microscopical (IEM) studies of skeletal and cardiac muscles that express titin isoforms. The previously deduced molecular properties of titin were used to model titins extensible region in the sarcomere as serially linked WLCs: rigid segments (containing folded Ig/Fn domains) and more flexible segments (PEVK segment). The model was tested on skeletal muscle fibers that express titin isoforms with tandem Ig and PEVK length variants. The model adequately predicts titins behavior along a wide sarcomere length range in skeletal muscle, but at long sarcome lengths (SLs), predicted forces are much higher than those determined experimentally. IEM reveals that this may result from Ig domain unfolding. Experiments were also performed on cardiac myocytes from mouse and cow that express predominantly a small cardiac titin isoform (N2B titin) or a large isoform (N2BA titin), respectively. The passive tension-SL relation of myocytes was found to increase more steeply with SL in mouse than in cow. IEM revealed an additional source of extensibility within both of these cardiac titins: the unique N2B sequence (absent in skeletal muscle). Furthermore, the PEVK segment of the N2BA isoform extended to a maximal length of approximately 200 nm, as opposed to approximately 60 nm for the N2B isoform. We propose that, along the physiological SL range, the long PEVK segment found in N2BA titins results in a low PEVK fractional extension and that this underlies the lower passive tensions of N2BA-expressing cow myocytes.

Poly-Ig tandems from I-band titin share extended domain arrangements irrespective of the distinct features of their modular constituents

The cellular function of the giant protein titin in striated muscle is a major focus of scientific attention. Particularly, its role in passive mechanics has been extensively investigated. In strong contrast, the structural details of this filament are very poorly understood. To date, only a handful of atomic models from single domain components have become available and data on poly-constructs are limited to scarce SAXS analyses. In this study, we examine the molecular parameters of poly-Ig tandems from I-band titin relevant to muscle elasticity. We revisit conservation patterns in domain and linker sequences of I-band modules and interpret these in the light of available atomic structures of Ig domains from muscle proteins. The emphasis is placed on features expected to affect inter-domain arrangements. We examine the overall conformation of a 6Ig fragment, I65–I70, from the skeletal I-band of soleus titin using SAXS and electron microscopy approaches. The possible effect of highly conserved glutamate groups at the linkers as well as the ionic strength of the medium on the overall molecular parameters of this sample is investigated. Our findings indicate that poly-Ig tandems from I-band titin tend to adopt extended arrangements with low or moderate intrinsic flexibility, independently of the specific features of linkers or component Ig domains across constitutively- and differentially-expressed tandems. Linkers do not appear to operate as free hinges so that lateral association of Ig domains must occur infrequently in samples in solution, even that inter-domain sequences of 4–5 residues length would well accommodate such geometry. It can be expected that this principle is generally applicable to all Ig-tandems from I-band titin.

Structural Analysis of B-Box 2 from MuRF1: Identification of a Novel Self-Association Pattern in a RING-like Fold†‡

The B-box motif is the defining feature of the TRIM family of proteins, characterized by a RING finger-B-box-coiled coil tripartite fold. We have elucidated the crystal structure of B-box 2 (B2) from MuRF1, a TRIM protein that supports a wide variety of protein interactions in the sarcomere and regulates the trophic state of striated muscle tissue. MuRF1 B2 coordinates two zinc ions through a cross-brace alpha/beta-topology typical of members of the RING finger superfamily. However, it self-associates into dimers with high affinity. The dimerization pattern is mediated by the helical component of this fold and is unique among RING-like folds. This B2 reveals a long shallow groove that encircles the C-terminal metal binding site ZnII and appears as the defining protein-protein interaction feature of this domain. A cluster of conserved hydrophobic residues in this groove and, in particular, a highly conserved aromatic residue (Y133 in MuRF1 B2) is likely to be central to this role. We expect these findings to aid the future exploration of the cellular function and therapeutic potential of MuRF1.