Protein–protein interactions between intermediate chains and the docking complex of Chlamydomonas flagellar outer arm dynein

Abstract

Outer arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association.

Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme

“…Third, DC1-2-3 rescued the phenotype of the oda3ida4 mutant (lacking the ODA-DC, OAD, and inner arm dyneins a, c, and d; nonmotile) when introduced by electroporation-mediated protein delivery: A few hours after introduction of proteins, ∼19% (n = 2,735) of cells became motile whereas all control cells (pulse applied without proteins) remained nonmotile (n = 862) (Movies S1 and S2). This motility recovery rate was much higher than when DC1-2 was used (<2%) (25), suggesting that DC3 may stabilize the ODA-DC. Finally, when DC1-2-3 was added to isolated axonemes of the oda1 mutant lacking the ODA-DC and OAD (Fig.…”

“…Recombinant ODA-DC (which is hereafter referred to as "DC1-2-3" to distinguish it from the native ODA-DC isolated from the axoneme) was prepared by combining bacterially expressed DC3 (20) with the DC1-DC2-6×His complex produced by coexpression of these proteins in cultured Sf21 insect cells (25), followed by purification on a Ni·NTA agarose column. The eluates contained DC1, DC2-6×His, and DC3, verifying that DC1-2-3 forms a single stable complex (Fig.…”

“…The ODA-DC molecules cooperatively bind to the doublet in an end-toend manner and establish a 24-nm periodicity. Subsequently, OAD binds to the outer doublet through its association with the ODA-DCs on the doublet, and this interaction is stabilized through the direct interaction of IC1 with the microtubule, thereby establishing the 24-nm periodicity in the OAD arrangement (24,25,29,30).…”

“…Defects in CCDC114 are known to cause PCD and the patients lack outer dynein arms, suggesting that DC2 functions in OAD docking in humans also (22,23). The ODA-DC binds OAD via at least three interactions: one between DC1 and the OAD intermediate chain IC1, one between DC1 and the other OAD intermediate chain IC2, and one between DC2 and the OAD light chain LC7b (24,25). However, we still do not understand the architecture of the ODA-DC and the detailed manner of its binding to the doublet microtubules, which will be essential for understanding the molecular basis of OAD periodicity.…”

Docking-complex-independent alignment of Chlamydomonas outer dynein arms with 24-nm periodicity in vitro

“…We hypothesized that docking complexes reinforce the interaction between the ODA and DMT because the docking complex has a higher affinity for DMTs when compared with the affinity for the intermediate chains of the ODA (Ide et al, 2013;Owa et al, 2014). To test this hypothesis, we increased the ionic strength of the buffer used for resuspension of the ODA-reconstituted oda3 axonemes from 50 mM CH 3 CO 2 K to 100 mM NaCl.…”