The structure of the EF-hand-GAR module of ACF7 and the delineation of microtubule binding determinants

The dynamic microtubule (MT) and F-actin networks of the cytoskeleton are in constant flux, undergoing structural rearrangement in order for the cell to perform complex, evolutionarily-invariant processes such as cellular migration, focal adhesion dynamics, dendrite formation, and intracellular trafficking. These processes dually engage both networks simultaneously and require strict control over both dynamics and polarity of these cytoskeletal elements. While these interactions have been shown to be integral for these cellular processes, this is an area of active research. Spectraplakins are evolutionarily conserved, giant proteins that can directly cross-link MTs and F-actin concurrently. This linkage has been shown to be instrumental for proper cellular rearrangement and polarization during many cellular processes. Loss of functional spectraplakins results in a variety of deleterious phenotypes. In vivo studies have shown that loss of ACF7 inhibits gastrulation, leading to pre-implantation lethality, while loss of BPAG1 causes early-onset postnatal death. The autoimmune disease bullous pemphigoid, which has characteristic persistent blistering, is caused by loss of epithelially expressed BPAG1. Loss of neuronal BPAG1 leads to the neurodegenerative disorder dystonia. In each of these disease states, cells exhibit major morphological disruptions in cytoskeletal structure and organization. Similarly, knocking out ACF7 or BPAG1 in cells produces defects in cellular polarization, migration, intracellular vesicular trafficking, and focal adhesion dynamics. Spectraplakins undoubtedly have a vital role in many core cellular processes. It has previously been determined that a minimal MT-binding domain consists of a GAR domain, though the binding mechanism remained poorly understood. The role of the juxtaposed EF-hands domain has also remained elusive, though it has been shown to be functionally required in both neuronal extension and path finding. Here, we present the 2.8 Å crystal structure of the EF1-EF2-GAR module. While these domains are tethered, they are connected by a flexible linker and are therefore considered independent domains. The EF1- EF2 module binds two Ca2+ molecules and has an overall prototypical EFβ fold. The GAR domain consists of a novel α/β-sandwich fold that coordinates zinc. We have also mapped the MT binding region of the GAR and EF1-EF2 domains to positive patches that lies distal to the Zn2+ and Ca2+ binding domains, respectively. We also provide evidence that while the GAR domain is the principal MT binding element the EF-hands of ACF7 directly bind MTs, enhancing the MT-binding ability of the cassette.Doctor of Philosoph