Structural and Biochemical Analysis of Dynein Light Chain-Mediated Homodimerization of Cytoskeletal and Nuclear Pore Proteins Public Deposited

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  • March 21, 2019
Creator
  • Romes, Erin MacKenzie
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
Abstract
  • The dynein light chain, Lc8/Dyn2, is a ubiquitous protein that acts as a scaffold, binding to many different target proteins in various cellular contexts. Here we describe S. cerevisiae Dyn2's biophysical and structural interaction with the dynein intermediate chain, Pac11 at the dynein complex, and also Dyn2's interaction with a nuclear pore protein, Nup159 in the cytoplasmic fibrils. We also demonstrate the structural and binding similarities between the Drosophila homolog, Lc8 binding to a centriole duplication protein, Ana2 and Dyn2's interaction with Pac11 or Nup159. We obtained the first high-resolution crystal structure of Dyn2 bound to Nup159 peptides and subsequent structures of a homodimer of Dyn2 bound to two identical peptides of Pac11, and a homodimer of Lc8 bound to two identical Ana2 peptides. We also characterized the thermodynamic binding profiles of Dyn2/Lc8 interacting with Pac11, Ana2, or Nup159 peptide binding sites and discovered that both Dyn2 and Lc8 are capable of two modes of binding peptides, endothermically or exothermically with KDs in the range of 0.5 to 20 μM. Results from these experiments highlight Dyn2/Lc8's ability to act as a dimerization machine to possibly optimize Pac11, Ana2 and Nup159's respective functions in the cell. Each of the Dyn2/Lc8 target proteins we have described here represents an essential component in their respective contexts. Pac11 is an essential scaffold that binds directly to the dynein motor chain to modulate dynein velocity and processivity through various binding interactions, Ana2 is an essential centriole duplication protein that is responsible for nucleating a single procentriole so that a cell does not experience genomic instability as a result of improper chromosome distribution, and Nup159 is an essential protein in regulating mRNA export out of the nucleus through the nuclear pore. We provide evidence that Dyn2/Lc8 interacting in each of these processes affords the target protein the ability to optimize through dimerization.
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  • In Copyright
Advisor
  • Slep, Kevin
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill
Graduation year
  • 2012
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