Mathias Gautel

Structural basis for activation of the titin kinase domain during myofibrillogenesis

The giant muscle protein titin (connectin) is essential in the temporal and spatial control of the assembly of the highly ordered sarcomeres (contractile units) of striated muscle. Here we present the crystal structure of titins only catalytic domain, an autoregulated serine kinase (titin kinase). The structure shows how the active site is inhibited by a tyrosine of the kinase domain. We describe a dual mechanism of activation of titin kinase that consists of phosphorylation of this tyrosine and binding of calcium/calmodulin to the regulatory tail. The serine kinase domain of titin is the first known non-arginine–aspartate kinase to be activated by phosphorylation. The phosphorylated tyrosine is not located in the activation segment, as in other kinases, but in the P+ 1 loop, indicating that this tyrosine is a binding partner of the titinkinase substrate. Titin kinase phosphorylates the muscle protein telethonin in early differentiating myocytes, indicating that this kinase may act in myofibrillogenesis.

Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction?

Cardiac myosin binding protein‐C (cardiac MyBP‐C, cardiac C protein) belongs to a family of proteins implicated in both regulatory and structural functions of striated muscle. For the cardiac isoform, regulatory phosphorylation in vivo by cAMP‐dependent protein kinase (PKA) upon adrenergic stimulation is linked to modulation of cardiac contraction. The sequence of human cardiac MyBP‐C now reveals regulatory motifs specific for this isoform. Site‐directed mutagenesis identifies a LAGGGRRIS loop in the N‐terminal region of cardiac MyBP‐C as the key substrate site for phosphorylation by both PKA and a calmodulin‐dependent protein kinase associated with the native protein. Phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform‐specific site. This phosphorylation switch can be mimicked by aspartic acid instead of phosphoserine. Cardiac MyBP‐C is therefore specifically equipped with sensors for adrenergic regulation of cardiac contraction, possibly implicating cardiac MyBP‐C in cardiac disease. The gene coding for cardiac MyBP‐C has been assigned to the chromosomal location 11p11.2 in humans, and is therefore in a region of physical linkage to subsets of familial hypertrophic cardiomyopathy (FHC). This makes cardiac MyBP‐C a candidate gene for chromosome 11‐associated FHC.

Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis

Skeletal muscle is the dominant organ system in locomotion and energy metabolism. Postnatal muscle grows and adapts largely by remodelling pre-existing fibres, whereas embryonic muscle grows by the proliferation of myogenic cells. Recently, the genetic hierarchies of the myogenic transcription factors that control vertebrate muscle development — by myoblast proliferation, migration, fusion and functional adaptation into fast-twitch and slow-twitch fibres — have become clearer. The transcriptional mechanisms controlling postnatal hypertrophic growth, remodelling and functional differentiation redeploy myogenic factors in concert with serum response factor (SRF), JUNB and forkhead box protein O3A (FOXO3A). It has also emerged that there is extensive post-transcriptional regulation by microRNAs in development and postnatal remodelling.

Towards a molecular understanding of titin.

Titin is at present the largest known protein (M(r) 3000 kDa) and its expression is restricted to vertebrate striated muscle. Single molecules span from M‐ to Z‐lines and therefore over 1 micron. We have isolated cDNAs encoding five distant titin A‐band epitopes, extended their sequences and determined 30 kb (1000 kDa) of the primary structure of titin. Sequences near the M‐line encode a kinase domain and are closely related to the C‐terminus of twitchin from Caenorhabditis elegans. This suggests that the function of this region in the titin/twitchin family is conserved throughout the animal kingdom. All other A‐band sequences consist of 100 amino acid (aa) repeats predicting immunoglobulin‐C2 and fibronectin type III globular domains. These domains are arranged into highly ordered 11 domain super‐repeat patterns likely to match the myosin helix repeat in the thick filament. Expressed titin fragments bind to the LMM part of myosin and C‐protein. Binding strength increases with the number of domains involved, indicating a cumulative effect of multiple binding sites for myosin along the titin molecule. We conclude that A‐band titin is likely to be involved in the ordered assembly of the vertebrate thick filament.

Mechanoenzymatics of titin kinase

Biological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM-based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation and substrate turnover.

Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is ∼800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3–like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH2-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca2+/calmodulin, as well as G protein–coupled signal transduction in the sarcomere.

Molecular structure of the sarcomeric Z-disk: two types of titin interactions lead to an asymmetrical sorting of α-actinin

The sarcomeric Z‐disk, the anchoring plane of thin (actin) filaments, links titin (also called connectin) and actin filaments from opposing sarcomere halves in a lattice connected by α‐actinin. We demonstrate by protein interaction analysis that two types of titin interactions are involved in the assembly of α‐actinin into the Z‐disk. Titin interacts via a single binding site with the two central spectrin‐like repeats of the outermost pair of α‐actinin molecules. In the central Z‐disk, titin can interact with multiple α‐actinin molecules via their C‐terminal domains. These interactions allow the assembly of a ternary complex of titin, actin and α‐actinin in vitro, and are expected to constrain the path of titin in the Z‐disk. In thick skeletal muscle Z‐disks, titin filaments cross over the Z‐disk centre by ∼30 nm, suggesting that their α‐actinin‐binding sites overlap in an antiparallel fashion. The combination of our biochemical and ultrastructural data now allows a molecular model of the sarcomeric Z‐disk, where overlapping titin filaments and their interactions with the α‐actinin rod and C‐terminal domain can account for the essential ultrastructural features.

The spectrin repeat: a structural platform for cytoskeletal protein assemblies

Spectrin repeats are three‐helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a ‘switchboard’ for interactions with multiple proteins with a more regulatory role. We describe the structure of the α‐actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The α‐actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin‐binding domains positioned at the two ends of the α‐actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z‐line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of α‐actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src‐homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.

Molecular structure of the sarcomeric M band: mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myomesin

The M band of sarcomeric muscle is a highly complex structure which contributes to the maintenance of the regular lattice of thick filaments. We propose that the spatial coordination of this assembly is regulated by specific interactions of myosin filaments, the M band protein myomesin and the large carboxy‐terminal region of titin. Corresponding binding sites between these proteins were identified. Myomesin binds myosin in the central region of light meromyosin (LMM, myosin residues 1506–1674) by its unique amino‐terminal domain My1. A single titin immunoglobulin domain, m4, interacts with a myomesin fragment spanning domains My4–My6. This interaction is regulated by phosphorylation of Ser482 in the linker between myomesin domains My4 and My5. Myomesin phosphorylation at this site by cAMP‐dependent kinase and similar or identical activities in muscle extracts block the association with titin. We propose that this demonstration of a phosphorylation‐controlled interaction in the sarcomeric cytoskeleton is of potential relevance for sarcomere formation and/or turnover. It also reveals how binding affinities of modular proteins can be regulated by modifications of inter‐domain linkers.

Molecular Basis for Cross-Linking of Actin Filaments: Structure of the α-Actinin Rod

We have determined the crystal structure of the two central repeats in the alpha-actinin rod at 2.5 A resolution. The repeats are connected by a helical linker and form a symmetric, antiparallel dimer in which the repeats are aligned rather than staggered. Using this structure, which reveals the structural principle that governs the architecture of alpha-actinin, we have devised a plausible model of the entire alpha-actinin rod. The electrostatic properties explain how the two alpha-actinin subunits assemble in an antiparallel fashion, placing the actin-binding sites at both ends of the rod. This molecular architecture results in a protein that is able to form cross-links between actin filaments.