This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The actin cytoskeleton of eukaryotic cells undergoes constant remodeling, resulting in various types of cytoskeletal structures, such as filopodia, lamellipodia, stress fibers and focal adhesions [1]. These dynamic structures play essential roles in many cellular functions, including motility, cytokinesis, fibroblast migration and endocytosis. Actin, a 42-kDa ATPase and the most abundant protein in eukaryotic cells, is the primary component of the cytoskeleton. Actin exists is two forms, a monomeric (G-actin) form and a filamentous (F-actin) form. F-actin is most commonly described as a double helix of head-to-tail interacting actin monomers [2]. The filament is structurally and kinetically asymmetric. In addition to nucleotide hydrolysis by actin, the time, location, association rate and specific type of cytoskeletal structure are determined by signaling proteins as well as by the actions of a vast number of actin-binding proteins (ABPs). The main goal of our research is to understand how protein-protein interaction networks bring together cytoskeleton scaffolding, nucleation and elongation factors to accomplish cellular functions. To achieve this goal we use a combination of structural and biophysical methods.
The aim of this proposal is to request X-ray data collection time to obtain high-resolution structures of macromolecular complexes of the actin cytoskeleton. Time is requested for data collection on the following projects:1. Study of a complex of -actinin and zyxin. -Actinin is as an antiparallel homodimer. Each subunit consists of an N-terminal actin-binding domain (ABD), composed of tandem calponin-homology (CH) domains, followed by four spectrin repeats, and a C-terminal calmodulin-like (CaM) domain. -Actinin was originally described as an actin-crosslinking protein, but it has now become evident that -actinin possesses an exceptionally large number of molecular partners, most of which are focal adhesion and dense bodies-associated proteins [3-5]. The interactions of -actinin with these partners are typically mediated by the spectrin repeat region [6]. The structure of this region (also known as the rod domain) has been determined and shown to form a twisted antiparallel dimer with a conserved acidic surface, which is thought to play a role in target recognition [7]. In particular, this region is believed to mediate the interaction of -actinin with the focal-adhesion protein zyxin. To understand this interaction, which could serve as a paradigm for many of -actinins interactions in focal adhesion complexes, we co-crystallized an -actinin-zyxin complex. The complex consists of the spectrin repeat dimer of -actinin and an N-terminal fragment of zyxin, containing a basic sequence, which is thought to bind in the acidic patch of the spectrin repeats. The total mass of the complex is ~120 kDa. We have obtained crystals in two different forms. The crystals diffract the X-rays to low resolution (~4 ) using out home source. We have many crystals of the two forms frozen in glycerol as cryo-protectant. The crystals were stored in liquid nitrogen. We now request data collection time to determine the high-resolution structure of this complex.2. Structure of the actin-binding domain of -actinin-4.Alpha-actinin-4 is ubiquitously expressed. In collaboration with the group of Martin Pollak at the Harvard Medical School, we are studying point mutations in ABD of -actinin-4 that cause autosomal-dominant kidney failure. We have crystallized these mutants and would like to request data collection time to determine their high-resolution structures. The structures are expected to reveal the molecular basis for the compromised interactions of the mutants with actin. Crystals, diffracting the X-rays to relatively high resolution are available, and have been frozen.3. Structure of the IMD of missing-in-metastasis with membrane phospholipids.Missing-in-metastasis (MIM) is a multi-domain adaptor protein that links extracellular signals to actin cytoskeleton remodeling. MIM contains independent F- and G-actin-binding domains, consisting respectively of an N-terminal 250-aa IMD and a C-terminal WH2. We have recently determined the crystal structure of this domain [8]. The structure is related to that of the BAR domain. Like the BAR domain, the IMD has been implicated in membrane binding. Yet, comparison of the structures reveals that the membrane-binding surfaces of the two domains have opposite curvatures, which may determine the type of curvature of the interacting membrane. To understand how the IMD senses and interacts with membrane phospholipids, we have examined the specificity and affinities of the IMD for different types of lipids using surface plasmon resonance (SPR). We have also co-crystallized the IMD with some of these lipids and would like to request data collection time to determine the structures. 4. Structure of the toxofilin-actin complex.Many human pathogens exploit the actin cytoskeleton of host cells for infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein involved in invasion.We are studying the interaction of toxofilin with actin using biophysical and structural approaches. Our studies indicate that toxofilin may bind up to three actin monomers. We have recently determined the crystal structure of toxofilin with actin in a 1:1 complex (Lee et al., in preparation). We have now also crystallized a 1:2 toxofilin:actin complex. We would like to request data collection time to determine this new structure. The structures will provide important insights about the toxofilin-actin interaction, which may be of medical significance to develop treatments against apicomplexan parasites.5. Conformational changes in Dictyostelium actin upon phosphorylation. Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores, which survive under starvation stress. At this stage, high levels of actin phosphorylation on Tyr-53 occur and correlate closely with rearrangements of the actin cytoskeleton and changes in cell shape [9, 10]. Tyr-53 phosphorylation substantially inhibits nucleation and elongation from the pointed ends of actin filaments and reduces the elongation from the barbed ends. To understand the molecular mechanism and actin conformational change upon phosphorylation, high-resolution structures of both the phosphorylated and unphosphorylated forms of Dictyostelium actin must be determined. In collaboration with Ed Korn at the NIH, we have crystallized these two phosphorylation states of Dictyostelium actin in complexes with both vitamin-D binding protein (DBP) and profilin. We request data collection time to determine the structures. References1. Pollard, T.D., and Borisy, G.G. (2003). Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453-465.2. Holmes, K.C., Popp, D., Gebhard, W., and Kabsch, W. (1990). Atomic model of the actin filament. Nature 347, 44-49.3. Otey, C.A., and Carpen, O. (2004). Alpha-actinin revisited: a fresh look at an old player. Cell Motil Cytoskeleton 58, 104-111.4. Broderick, M.J., and Winder, S.J. (2005). Spectrin, alpha-actinin, and dystrophin. Adv Protein Chem 70, 203-246.5. Zaidel-Bar, R., Cohen, M., Addadi, L., and Geiger, B. (2004). Hierarchical assembly of cell-matrix adhesion complexes. Biochem Soc Trans 32, 416-420.6. Djinovic-Carugo, K., Gautel, M., Ylanne, J., and Young, P. (2002). The spectrin repeat: a structural platform for cytoskeletal protein assemblies. FEBS Lett 513, 119-123.7. Ylanne, J., Scheffzek, K., Young, P., and Saraste, M. (2001). Crystal structure of the alpha-actinin rod reveals an extensive torsional twist. Structure 9, 597-604.8. Lee, S.H., Kerff, F., Chereau, D., Ferron, F., Klug, A., and Dominguez, R. (2007). Structural Basis for the Actin-Binding Function of Missing-in-Metastasis. Structure 15, 145-155.9. Kishi, Y., Clements, C., Mahadeo, D.C., Cotter, D.A., and Sameshima, M. (1998). High levels of actin tyrosine phosphorylation: correlation with the dormant state of Dictyostelium spores. J Cell Sci 111 (Pt 19), 2923-2932.10. Liu, X., Shu, S., Hong, M.S., Levine, R.L., and Korn, E.D. (2006). Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments. Proc Natl Acad Sci U S A 103, 13694-13699.
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