Structure and Regulation of the Myofibrillar Z-disc Interactome

Mature Z-discs are composed of a plethora of different proteins, and they may be regarded as one of the most complex macromolecular structures in biology. This intricate web of interactions needs to be continuously finely tuned and maintained. Recent studies have suggested an important role of signalling pathways in regulating the dynamics and turnover of Z-disc components and of chaperone-assisted selective autophagy (CASA) in the maintenance of Z-disc structure and function. In the second phase of this project we aim to address two main questions: (1) How does phosphorylation modulate selected protein-protein interactions of Z-disc complexes, in particular those of filamin C with its interaction partners? (2) How does myopodin/SYNPO2 mediate interactions with the autophagosome formation sub-complex of the CASA machinery? Both projects will be performed employing integrative structural biology approaches, in which high resolution structural studies are complemented by lower-resolution structural biology approaches to generate valuable structural information. Structural analyses will subsequently be the basis for designing experiments addressing functional questions using a blend of biochemical and cellular assays.

Movement is vital to all living organisms, from the transport of cellular organelles to the movement of entire organisms. Sarcomeres are the smallest contractile units of striated skeletal and heart muscles. Z-discs - the boundaries between the adjacent sarcomeres play pivotal roles in sustaining sarcomeric architecture, signalling, mechanosensing, and protein homeostasis. Mature Z-discs contain upwards of 50 different proteins and are regarded as one of the most complex macromolecular structures in biology. -Actinin is the major component of the Z-disc, where it cross-links actin filaments from neighbouring sarcomeres. Filamin C not only cross-links actin filaments but acts also as a mecanosensor for the force exerted on the Z-discs. The complex Z-disk interactome is further tuned by post-translational modifications, the best studied are phosphorylations. Our studies focused on the understanding of the impact of posttranslational modifications in filamin C domains 19-21, on the interaction with selected binding partners (FATZ-1 and Pgm5/aciculin), structural analysis of complexes of FATZ-1 with filamin C and -actinin-2, and on the structural and biochemical analysis of PGM5/aciculin. Interestingly, found that phosphorylations notably increase the capacity of the filamin C to refold and we hypothesize that this property might be important for the increased capacity of the protein to recover from mechanical strain and mechanical damage during muscle contraction. We also found that negatively phosphorylation regulates filamin C interactions with FATZ-1. Our structural studies revolved around complexes of FATZ-1 with filamin C and -actinin-2 and phosphoglucomutase -5/aciculin, that was recently discovered to bind to Z-disk proteins. We determined the crystal structures of FATZ-1 in complex with filamin C (domain 21) and with -actinin-2 (central rod domain). These are the first crystal structures of intrinsically disordered Z-disk protein FAZT-1 with two of its major binding partners, which shed light on how these proteins interact at the atomic level. Furthermore, our biochemical and structural analysis of phosphoglucomutase 5 (PGM5)/aciculin showed that the protein has enzymatic activity and that the active site is structurally different from canonical phosphoglucomutases. These results change the paradigm regarding PGM5/aciculin role in the Z-disks from a purely structural protein to a scaffolding enzyme potentially regulated by interaction partners.