Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

Amanda E Siglin,Tatyana Polenova,Jeffrey K Moore,Sarah Tan,John A Cooper,Shangjin Sun,James D Lear,John C Williams,Martin Poenie,Eugene A Permyakov

doi:10.1371/journal.pone.0059453

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 p150Glued; 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 p150Glued. 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 p150Glued 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 p150Glued 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 p150Glued. 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.

Highlights

Regulated, vectorial transport of signaling molecules, membranous organelles and cytoskeletal elements as well as the generation of forces to separate chromosomes and drive cellular motility are critical for cell function, viability and division [1]
When comparing our circular dichroism (CD) and analytical ultracentrifugation (AUC) data to recent electron microscopy images (See Fig. 1C in Imai [43]), we propose that CC1A constitutes the ‘‘arm’’ of the dynactin complex
The dynein-dynactin interaction has been shown to be critical for organelle transport, cytoskeletal organization and cell division [2,3,17]

Summary

Introduction

Vectorial transport of signaling molecules, membranous organelles and cytoskeletal elements as well as the generation of forces to separate chromosomes and drive cellular motility are critical for cell function, viability and division [1]. Previous reports indicate the p150Glued binding site on the dynein IC spans residues 1 to 106 of the IC (IC2C isoform) [11,12,13] and recent NMR results have further refined the dynactin interface to residues 1–87 [21] This region contains a highly predicted coiled-coil, splice sites found in mammalian dynein [22], and a serine rich region. We further confirm the importance of these interactions in vitro with cell-based assays These data suggest that residues 10–44 of the Nterminus of the dynein IC are sufficient to bind p150Glued in a predominantly electrostatic interaction and that in doing so, they undergo a disordered-to-order transition. They highlight the functional aspects of coupling disordered regions through a bivalent-bivalent interaction and provide additional support for a regulatory role of the LCs on dynein function [25,26,27]

Materials and Methods

Results

Findings

Discussion