Structural basis of a-actinin-titin filaments interactions

The principle components of striated muscle sarcomeres include parallel arrays of actin-containing thin filaments that span the I-band and overlap with myosin-containing thick filaments in the A-band. The third filament system is formed by single molecules of titin (the largest vertebrate protein identified to date), which span half sarcomeres. A major challenge in understanding the networks of interactions that are involved in function and control of the Z- disc in a striated muscle is the definition of and understanding the molecular basis of protein-protein interactions. The modular nature of alpha-actinin and titin allows for multiple protein interactions to occur simultaneously, suggesting that they may act as part of a nexus for the assembly of the Z-disc complex. Although a series of the interactions occur among Z-disc proteins, and a body of structural information is already available on individual members or their domain, not much is known about the overall architecture of the interacting components at molecular detail, and both functional and structural implications of these interactions remain largely unknown. The principal objective of the proposed research is to investigate the protein-protein assemblies that connect the titin filament to alpha-actinin in the Z-disc at the molecular/structural level. We aim at generating macromolecular complexes centered on two major players, Z-disk alpha-actinin and telethonin. Binary and higher complexes with interacting partners or domains of interacting partners (MLP, ZASP, FATZ, -filamin) will be produced for structural analysis.

Movement is vital to all living organisms, from the transport of single molecules in cells to the movement of the entire organism. In vertebrates, skeletal muscle is essential for all voluntary movements such as walking, running, swimming or flying. Involuntary movements, such as those occurring in cardiac muscle resulting in contraction of the heart (beating), or those occurring in smooth muscle resulting in peristalsis are fundamental to the viability of the organisms. Sarcomeres are the smallest cellular unit behind the operation of skeletal and heart muscles. Furthermore, dysfunctional sarcomeres are responsible for a long list of diseases that reduce the quality of life and burden the health care sector throughout the world. One of the functionally most complex sub-compartments of the sarcomere is the Z-disk that forms the lateral boundaries between adjacent sarcomeres in which the antiparallel actin filaments from adjacent sarcomers are cross-linked by alpha-actinin, which in turn binds to molecular ruler titin. The intriguing attraction and challenge of the Z-disk is the multiplicity of the protein-protein interactions. In this project we set for structural characterisation of selected Z-disk proteins, in particular on alpha-actinin, myotilin and the interaction between titnin and obscurin, using as the primary structural biology technique macromolecular crystallography, complemented by solution small angle X-ray scattering and FRET techniques. Crystal structure of human titin`s C-terminal domain M10 (residues 34253-34350) and the N-terminal domain (residues 1-105) of obscurin-like 1 protein clearly explained the basis of the mechano-lability of this complex, expected for its function as a mechanosensor. The small angle X-ray scattering based model of the dimer of human myotilin immunoglobulin like domains showed the molecular basis of the ability of the myotilin to not only bind but also bundle actin filaments. The crystal structure of human alpha actinin-2 displays the molecular architecture of an antiparallel dimer, which is the molecular basis for its ability to cross-link antiparallel actin filaments from adjacent sarcomeres. Detailed structural analysis furthermore explains the structural basis for its regulation PIP2 which promotes alpha-actinin binding to titin and consequently targeting its to Z-disk.