Structural studies on protein scaffolds related to muscle physiology and disease : the titin filament, its associated component MuRF-1 and nuclear LAP2[alpha]

The titin molecule has a length of overµm and functions as a colossal protein scaffold in the muscle sarcomere. Up to 90% of its total mass is composed of repetitive immunoglobulin (Ig) and fibronectin (FnIII) domains that form linear tandems interspersed by unique sequences, among them a Ser/Thr kinase domain located at its C-terminus. The distinct pattern of Ig and FnIII motifs N-terminal to the kinase domain is conserved in other „giant kinases“ and invertebrate titin homologues. In vertebrate titin, it is involved in the specific recruitment of the ubiquitin ligase MuRF-1 to the filament. MuRF-1 is involved in the pathological atrophy of skeletal and cardiac muscle. We have determined the crystal structure of titin A168-A170 comprising two Ig and one FnIII domains and established its binding to MuRF-1 in solution. We analysed the structure with the aim to understand the interdomain relationships between repetitive Ig and FnIII subunits in titin as well as to shed light into the molecular determinants that confer specificity to ligand binding on the scaffold and in particular in the M-line interface to MuRF-1. A168-A170 shows an extended, rigid architecture. Its surface displays a shallow groove along its full length as well as a unique loop protrusion, both features conceivably mediating MuRF-1 binding. Moreover, our ITC data show that binding occurs with high affinity between residues 166-315 of MuRF-1. These data suggest that A168-A170 is of interest to attempt therapeutic inhibition of MuRF-1-mediated muscle turnover. In addition we have elucidated the structure of the B-box domain of MuRF-1 to further investigate the role of MuRF-1 in homo- and hetero-oligomeric interactions at the M-line region. We found that MuRF-1 B-box adopts a RING-finger-like fold and exists in a dimeric state in solution. The domain possesses characteristic surface properties that are likely to mediate interactions of MuRF-1 with other sarcomeric components that are important in MuRF-1 function at the M-line. Finally, we have also carried out the biophysical characterization of the nuclear adaptor protein LAP2! that interacts with the nuclear lamina scaffold. Conceptually, LAP2! and the nuclear lamina are closely related systems to MuRF-1 and titin. This characterization, whose ultimate finality is to understand the interaction of LAP2! with lamin A/C establishes now the basis for a future structure elucidation.
This work illustrates how scaffold protein systems, which are structural skeletons
composed of multiple repetitive units, can become functionalized by the recruitment of
specific shuttle proteins to their surface. Specific binding in such systems involves
steric factors as well as the evolution of unique sequence inserts at defined locations.
Recruited proteins often act as adaptors that, in turn, attract other cellular components.
They often result in large, heterogeneous molecular assemblies that amplify the
physiological response. In the case of titin, the potential formation of a signalosome
assembly at its M-line, surrounding a kinase domain, is thought to mediate mechanotransduction
pathways involved in the regulation of myofibril turn-over and, thereby, in
the adaptative remodelling of muscle to mechanical load.