Light Chain-dependent Self-association of Dynein Intermediate Chain

Abstract

Dynein light chains are bivalent dimers that bind two copies of dynein intermediate chain IC to form a cargo attachment subcomplex. The interaction of light chain LC8 with the natively disordered N-terminal domain of IC induces helix formation at distant IC sites in or near a region predicted to form a coiled-coil. This fostered the hypothesis that LC8 binding promotes IC self-association to form a coiled-coil or other interchain helical structure. However, recent studies show that the predicted coiled-coil sequence partially overlaps the light chain LC7 recognition sequence on IC, raising questions about the apparently contradictory effects of LC8 and LC7. Here, we use NMR and fluorescence quenching to localize IC self-association to residues within the predicted coiled-coil that also correspond to helix 1 of the LC7 recognition sequence. LC8 binding promotes IC self-association of helix 1 from each of two IC chains, whereas LC7 binding reverses self-association by incorporating the same residues into two symmetrical, but distant, helices of the LC7-IC complex. Isothermal titration experiments confirm the distinction of LC8 enhancement of IC self-association and LC7 binding effects. When all three light chains are bound, IC self-association is shifted to another region. Such flexibility in association modes may function in maintaining a stable and versatile light chain-intermediate chain assembly under changing cellular conditions.

Intrinsic Disorder in Dynein Intermediate Chain Modulates Its Interactions with NudE and Dynactin

“…IC is a 74-kDa protein with a predominantly unstructured N-terminal domain (15) and a WD-rich C-terminal domain. Within the predominantly unstructured N-terminal domain of Drosophila melanogaster IC, residues 3-36 are helical (1), consistent with the coiled-coil secondary structure predicted for residues 1-40 from sequence analysis (16), residues 48 -60 have nascent helical propensity (1), and residues 222-231 constitute a self-association domain (17). The N-terminal domain binds all three light chains.…”

“…They do not include the weak self-association domain (IC residues 222-231) (17), which is considerably distant from the residues involved in direct interaction with NudE and p150…”

Polybivalency and disordered proteins in ordering macromolecular assemblies

“…Binding enhancement arising from bivalency/polybivalency has been demonstrated in assembly of Swallow/LC8 complex [7, 8], the nuclear pore protein Nup159/LC8 complex [9] and the dynein intermediate chain/light chains complex [4, 5, 10, 11]. A common feature among the three systems is that a segment of their disordered regions has one or more recognition motifs for the hub protein LC8 [12].…”

Structural Dynamics and Multiregion Interactions in Dynein-Dynactin Recognition

“…Isotopically labeled 15 N and 13 C, 15 N proteins for NMR studies were prepared using published protocols (25,26). Protein expression and purification under native conditions were performed as described previously (27,28).…”