Alpha Actinin, Filamin C and their Complexes with Binding Partners

Sarcomers are building units of striated muscles, composed of highly ordered thick (myosin-based) and thin (actin- based) filaments that slide past each other during contraction. One of the functionally most complex sub- compartments of the sarcomere is the Z-disc that forms the lateral boundaries between adjacent sarcomeres. This region plays a central role as the site organizing thick filaments and titin into the molecular machinery that is required for muscle contraction. Apart from its major components - actin filaments, a-actinin and titin, Z-disc comprises a number of other proteins and acts as a platform for an enormously complex and dynamic web of interactions. The aim of the proposed research is to generate novel structural information at the molecular level on selected complexes centered on two essential sarcomeric Z-disc components: a-actinin and filamin C and two adaptors binding partners myopodin, and calsarcin/FATZ/myozenin, that both bind to a-actinin and filamin C and are in parallel being functionally characterized by partner groups (P1, P2, P10). Adaptor proteins play an important role in cytoskeletal assemblies and signalling by physically connecting proteins with distinct functions. We plan to characterize firstly binary interactions and subsequently study ternary and higher complexes in order to generate models about how adaptor proteins bind simultaneously to multiple ligands. We will employ a combination of structural and biophysical methods to characterize these interactions. To complement high and lower structural techniques (crystallography, NMR, small angle X-ray scattering) and well established binding experiments (ITC, SPR), we will use mass spectrometry techniques to characterise the interaction between the partners and inform the lower resolution structural and functional/mutational studies. The structural characterization will be correlated to the in vivo and in vitro functional data generated by the network partners. The research will therefore be performed in close collaboration with cell biology (P1, P2, P10), proteomics (P4), biophysics and structural biology groups of the network (P5, P7, P8, P9), at various levels from the fine design of functional constructs and their functional analysis in vivo and in vitro to the proteomics analysis of the Z-disc interactome.

We report here the characterization of myopodin cardiac muscle isoform in complex with synemin or VPS18 or a-actinin-2 using a combination of structural biology techniques - macromolecular crystallography, nuclear magnetic resonance and biochemical assays. Taken together, the results shade light on the role of different proteins in the process of Z-disk assembly and autophagy. We further report structural information generated on a-actinin-2 regulation and calsarcin/FATZ/myozenin - a-actinin-2 interaction, as well as structural and biophysical analysis of selected filamin C domains mutants involved in MFM mutations, which give molecular basis of the protein aggregation manifested in the diseased patients. 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 a-actinin, which in turn binds to molecular ruler titin and many other Z-disk proteins. The intriguing attraction and challenge of the Z-disk is the multiplicity of the protein-protein interactions. In this project we set for structural characterization of selected Z-disk proteins, in particular on myopodin and its interaction with other proteins involved in the molecular mechanism of mechanosensing in striated muscle.