Structural and functional studies of cardiac myosin binding protein - C and Cofilin

Abstract

Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in sarcomeric proteins, including cardiac myosin binding protein-C (cMyBP-C). The mechanisms by which mutations alter sarcomeric function are poorly understood. Cofilin is an important regulator of actin polymerisation within cells, especially in cancer, but the structure of the actin-cofilin complex is unknown. Aims of this thesis: 1) Construct fragments of cMyBP-C and it’s FHC mutants to assess their structural and functional roles, and, 2) Design cofilin mutants suitable for selective fluorescent labelling to assess the quaternary structure of the actin-cofilin complex. The cMyBP-C fragment examined was C1 + linker (C1-L) domains. C1 and C2 are immunoglobulin domains, with a phosphorylatable linker joining them. The linker structure was examined using homology modelling and nuclear magnetic resonance spectroscopy, which showed the presence of α-helix and a significant amount of random coil, thus demonstrating a lack of tertiary structure in the linker region. Functional analysis of C1-L and its FHC mutant constructs showed it binds to both F-actin and myosin with a similar affinity to the full C1-C2 construct, demonstrating that the C2 domain makes little contribution to binding. Additionally, FHC mutations result in reduced binding to F-actin, suggesting that these mutations may inhibit normal physiological interactions with F-actin during the contractile cycle. Cofilin regulates the cytoskeleton by binding and severing F-actin, but the molecular basis for these functions is poorly understood, due to the lack of a quaternary structure. We tested a recent computational model, by substituting mutant residues into cofilin for labelling with spectroscopic probes. The structure of cofilin was very sensitive to mutagenesis, especially at the N-terminus, which impaired actin binding. Inter-molecular distance measurements between actin and cofilin, using fluorescence resonance energy transfer (FRET) spectroscopy, provided strong confirmation for the proposed computational model of the cofilin-F-actin complex.