title: Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.
abstract: Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150(Glued); however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued). Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued) form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued) fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued). Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Siglin
authors.forename: Shangjin
authors.initials: S
authors.lastname: Sun
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Moore
authors.forename: Sarah
authors.initials: S
authors.lastname: Tan
authors.forename: Martin
authors.initials: M
authors.lastname: Poenie
authors.forename: James D
authors.initials: JD
authors.lastname: Lear
authors.forename: Tatyana
authors.initials: T
authors.lastname: Polenova
authors.forename: John A
authors.initials: JA
authors.lastname: Cooper
authors.forename: John C
authors.initials: JC
authors.lastname: Williams
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D056445:Cytoplasmic Dyneins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072159:Dynactin Complex; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: C000606360:DCTN1 protein, human; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D056445:Cytoplasmic Dyneins