Structural characterization and interaction studies of myopodin isoform A with Z-disc binding partners

Bezerra Eduardo Henrique Salviano
2015 unpublished
Myopodin is a member of the Podin family, a group of proteins widely expressed in muscle cells. Expression of myopodin has been observed at the early stages of the myofibrillogenesis process. In differentiated muscle cells myopodin is extensively found in the Z-discs, where it associates with several other fundamental components of the Z-disc, such as actin, α-actinin 2 and filamin C. Myopodin interacts with these proteins mostly through its central domain, a region conserved for all myopodin
more » ... oforms. The longest isoform of myopodin expressed in cardiomyocytes, myopodin A, possesses a PDZ domain at the N-terminus. This region of myopodin is responsible for its binding to the C-terminus of α-actinin-2, intermediate filament protein synemin and VPS18. The main aim of my thesis was to structurally and biochemically characterize the binding of myopodin to the selected binding partners and thus understand the function of myopodin in muscle cells. In this respect, by using ITC, I was able to measure differences in the affinities between PDZ domain of myopodin and C-terminal peptide of synemin, VPS18 and α-actinin 2. These complexes were successfully crystallized and their structures solved non-conventional binding of the peptide to the myopodin's PDZ has been observed in all structures. In addition, the data obtained from X-ray structure models of the individual complexes helped to uncover the structural determinants responsible for different binding affinities of the studied binding partners. Furthermore, while studying the biochemical properties of myopodin with SEC-MALLS at reducing and oxidizing conditions, differences in oligomeric state of the myopodin were observed. As this suggests, the actin bundling activity of myopodin central domain is achieved by two independent F-actin binding sites. We observed a dissociation constant of 2.0 µM using MST experiments for myopodin central domain and filamin C Ig d18-21 complex. However, it was suggested that phosphorylation of three serine sites of myopodin central domai [...]
doi:10.25365/thesis.38146 fatcat:bzgeh6zt2zeclevhlrg6vtzyme