Structure Study of Myosin Filament, Z-Disc and HIV Envelope Protein

We describe a cryo-electron microscopy 3D image reconstruction of relaxed myosin II-containing thick filaments from the flight muscle of the giant waterbug Lethocerus indicus. The relaxed thick filament structure is a key element of muscle physiology because it facilitates the re-extension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement in order to drive contraction. Previous models predicted that Lethocerus myosin in thick filament was unique in having an intermolecular head-head interaction, as opposed to the intramolecular head-head interaction observed in all other species. In contrast to the predicted model, we find an intramolecular head-head interaction, which is similar to that of other thick filaments but oriented in a distinctly different way. The arrangement of myosin's long α-helical coiled-coil rod domain has been hypothesized as either curved layers or helical sub-filaments. Our reconstruction is the first report having sufficient resolution to track the rod α-helices in their native environment at resolutions ~5.5Å, and shows that for Lethocerus, the layer arrangement is correct. Threading separate paths through the forest of myosin coiled-coils are four non-myosin peptides. We suggest that the unusual position of the heads and the rod arrangement separated by non-myosin peptides is an adaptation for mechanical signal transduction whereby applied tension disrupts the myosin heads as a component of stretch activation (Chapter 2). We improved the map resolution to 4.3Å of relaxed myosin filament and built an atomic model for the entire myosin molecule. In addition to the asymmetry of the heads, the entire coiled-coil lacks symmetry. The coiled-coil is broken in two places, once near the head-rod junction and again at the Skip 4 location. Unique interactions between the regulatory light chain and the myosin rods appear to stabilize the unique orientation of the myosin heads (Chapter 3). The Z-disk is a complex structure comprising over 40 proteins that are involved in the transmission of force developed during muscle contraction and in important signaling pathways that govern muscle homeostasis. In the Z-disk antiparallel thin filaments from adjacent sarcomeres are crosslinked by α-actinin. The structure of the Z-disk lattice varies greatly throughout the animal kingdom. In vertebrates the thin filaments form a tetragonal lattice, whereas invertebrate flight muscle has a hexagonal lattice. A detailed description at a high resolution of the Z-disk lattice is needed in order to better understand muscle function and disease. The molecular architecture of the Z-disk lattice in honey bee (Apis mellifera) is known from plastic embedded thin sections to a resolution of 7 nm, which is not sufficient to adequately dock known protein crystal structures. We have chemically isolated intact honey bee Z-disks from honey bee indirect flight muscle, thus obviating the need of plastic sectioning. We have employed cryo electron tomography and image processing to investigate the arrangement of proteins within the hexagonal lattice of the Apis Z-disk (Chapter 4). HIV envelope protein (Env), which consists of three gp120 and three gp41 molecules, recognizes the CD4 protein in T cells and facilitates fusion of HIV and T cell membranes. Env neutralization is a key step to prevent HIV infection. We investigate the binding effect of PG16 Fab to HIV-MN Env spikes in situ, by using cryo-electron tomography combined with subvolume alignment and classification to identify antibody-bound and antibody-free conformations of Env. By not biasing the spike selection, we find that Env spikes in situ do not have a distinctly symmetric trimeric structure, but can vary between a symmetric trimer and a T-shaped trimer. PG16 Fab changes the equilibrium towards a more symmetric trimer structure despite binding with a stoichiometry of one Fab per Env trimer (Chapter 5).