Cytoplasmic dynein is a microtubule-based molecular motor that uses the energy derived from ATP hydrolysis to move and transport cellular cargo towards the minus ends of microtubules. This transport is essential in several aspects of cell behavior including cell migration and division, chromosome segregation, and vesicle trafficking. The dynein heavy chain subunits bind to microtubules and contain the ATP sites that provide the motive force, while the light and intermediate chains are thought to bind to cargo and regulate the activity of the motor. Recent data from the Barbar lab have suggested that the role of light chain LC8 is to facilitate dimerization of the natively disordered N-terminal domain of dynein intermediate chain, rather than to primarily function as a cargo adaptor, as commonly thought. Light chain Tctex-1, which is a structural homolog of LC8 and binds the intermediate chain at a site contiguous to the LC8 binding site, is likely to have a similar role as LC8. The research project will test the hypothesis that the tandem roles of light chains LC8 and Tctex-1 are to promote dynein assembly and the interaction with the cargo adaptor dynactin, and not to act as cargo adaptors themselves. Using a combination of biophysical approaches including nuclear magnetic resonance spectroscopy (NMR), isothermal titration calorimetry and X-ray crystallography, the Barbar lab will elucidate the mechanisms of three important outcomes of the interactions of both light chains to dynein intermediate chain: 1) how they contribute to formation of a tight dynein assembly, 2) how they promote dimerization of the intermediate chain, and 3) how they may promote a tighter interaction of the intermediate chain with p150Glued subunit of dynactin. The essential roles of these light chains will be tested in vivo in the lab of Professor Tom Hays, a collaborator on this project. The studies should provide novel insights into the structural biology and thermodynamics of dynein motor function, and will complement the cell biological approaches that predominate in the field of intracellular transport. Since these studies provide the first structural insights into the role of the disorder-to-order transition in dynein assembly and its association with dynactin, they allow the opportunity to develop a tractable system to probe the role of intrinsic disorder in the assembly of large macromolecular complexes. Broader Impacts: The PI has a strong record of research contributions with undergraduate students, and of mentoring minorities and students with disabilities. The PI is taking a leading role on her campus to introduce molecular biophysics to the research community by providing access to instrumentation and training workshops. Furthermore, the PI will take an active role to promote basic science among high school students by providing opportunities for research lab experience, and by developing instructional material and a hands-on module in the Saturday Academy program. The latter is a highly successful program at Oregon State University for outreach for high school students in which biochemistry and biophysics have not yet been represented.
Cytoplasmic dynein is a microtubule-based molecular motor that uses the energy derived from ATP hydrolysis to transport cellular cargo. Transport by dynein motors is essential in numerous cellular processes, including chromosome separation during mitosis, cell migration, and movement of vesicles containing nutrients and other products. Dynein is a protein complex consisting of a motor domain and a number of smaller proteins known as the light and intermediate chains. Since there is a single gene for the motor domain, the versatility of dynein function is attributed to the more variable smaller proteins and their interactions with regulatory proteins. Binding of regulatory proteins causes conformational changes in the protein complex which affect the attachment of the motor to its cargo and the movement of the motor along the microtubule. The intermediate chain IC is central to dynein function because it connects the light chains that bind at its N-terminal domain with the heavy chain that binds at its C-terminal domain, and also includes the domain to which most known regulators bind. Protein molecules require a unique specific shape to recognize other proteins and do their biological function. What is intriguing about IC is that it does not have a specific shape but belongs to a special class of proteins referred to as intrinsically disordered proteins (IDPs). These proteins have multiple shapes and fluctuate quickly among these shapes depending on alterations in environmental or cellular conditions. IDPs play diverse roles in regulation of function in various binding partners, and are themselves highly amenable to regulation through post-translational modification. We have solved the high resolution structures of the three dynein light chains in complex with IC, and characterized their binding thermodynamics, which together proposed a novel role of protein disorder in dynein assembly. The mode of assembly of intrinsically disordered N-IC with dynein light chains defines a new class of disordered complexes. N-IC contains several disordered linear motifs that adopt unique structures when bound to dynein light chains. These induced structures complete the fold of the binding partners, while the linkers connecting the multiple linear motifs and not involved in binding remain disordered. The motif sequences in dynein IC have a propensity to fold either as b-strands (recognitions motifs for Tctex1 and LC8) or a-helix (recognition motif for LC7), but only adopt this stable secondary structure when bound to and incorporated into the fold of their respective partners. Quantitative binding studies on the complex show that binding of a light chain at one site of IC enhances binding to another light chain or IC self-association at a distant site. This process involves incorporation of two monovalent disordered protein chains (IC) into a complex in which the pair of disordered chains become a bivalent or polybivalent scaffold. Thus assembled IC remains elongated and flexible but forms a polybivalent scaffold that is modulated by long range coupling between IC self-association and light chains binding. We coined the term poly-bivalent scaffold to describe IC: bivalent because when the first dimeric light chain binds two IC chains it creates a bivalent IC to which the second light chain binds with higher affinity than it does to monovalent IC, and poly, because IC has several sites for binding bivalent ligands. A poly-bivalent system provides a stable assembly even when binding of any single ligand is moderate to weak. This type of scaffold may be general for assembly of disordered proteins. We have extended the assembly studies to dynein regulation by dynactin and NudE. What is intriguing about the dynein/dynactin/NudE interplay is that the two dynein regulators bind to the same segment of disordered IC but dynactin binds to an additional disordered segment. By manipulating the length, and chemical modification of this additional disordered segment, one regulator can be selected over the other even when both are present in the same cellular compartment. The impact of these studies are in the suggestion that the dynamic assembly of the dynein cargo attachment complex and the disorder retained in the complex allow for signal transmission to the motor domain upon binding of regulatory proteins.This integration of multidisciplinary approaches including NMR spectroscopy for atomic level interactions and thermodynamics is making a significant impact on the dynein field, given the complementary nature of our approaches to the cell biology and genetics data on which the field has primarily been established. Our experiments also advance the development of methods to probe the role of intrinsic disorder in assembly and regulation of large macromolecular machines in general. This proposal has provided opportunities for undergraduate and graduate students from minority groups to develop research skills in the Barbar laboratory, and has impacted progress in other disciplines by lending biophysical and structural expertise to microbiologists, plant pathologists and engineers at the OSU campus.
