Cryo‐EM Structures of Outer‐arm Dynein Array Bound to Microtubule Doublet Reveal a Mechanism for Motor Coordination

Eukaryotic cilia are evolutionarily conserved organelles that are responsible for cellular motility, sensory reception, embryonic development and intercellular communication. A typical motile cilium is characterized by its ʹ9 + 2ʹ scaffold, composed of nine microtubule doublets (MTDs), and a central pair complex (CPC). Two rows of axonemal dyneins, the outer‐arm dynein (OAD) and inner‐arm dynein (IAD), power the ciliary beating by sliding two adjacent MTDs. OAD is the key motor protein that generates the majority of mechanical forces required for this fundamental cellular process. A complete OAD is ~1.5‐2 megadalton in size and contains two (mammals) or three (in ciliates and algae) heavy chains (HCs), two intermediate chains (ICs) and a variety of light chains (LCs). Each OAD is divided into a head region that contains AAA+ rings for ATP hydrolysis and a tail that holds together the whole complex. The tail is permanently attached to MTD A‐tubule, while the head region of each HC contains a microtubule‐binding domain (MTBD) which binds and releases MTD B‐tubule depending on the nucleotide‐binding states. Thousands of OADs are arrayed in the axoneme to drive a rhythmic ciliary beat, which is required to OADs locally synchronize their conformations and coordinate with each other. Current understanding is mainly based on cryo‐electron tomography (cryo‐ET) which suggests that adjacent OADs are indirectly connected via a series of linker structures. Nevertheless, it is largely unclear how the OAD arrays are formed and why the array is important for ciliary beat. Furthermore, the mechanism underlying the motor coordination remains elusive. We set out to reveal the mechanism of motor coordination to shed lights on how the conformational changes of arrayed OAD are linked to force generation and beating propagation. We use the model system T. thermophila for biochemical and cryo‐EM analysis. T. thermophila OAD is the first discovered dynein, which contains three HCs (α‐, β‐ and γ‐HC), two ICs (IC2 and IC3) and a set of indefinite LCs. We reconstitute the purified OADs onto MTD to mimic the native OAD arrays and determined the structures of free OAD and OAD arrays bound to MTDs (OAD‐MTD) in two different microtubule‐binding states (MTBS) by cryo‐electron microscopy (cryo‐EM). We show that microtubule binding induces free OADs to spontaneously adopt a parallel conformation, which is primed for array formation in a tail‐to‐head (TTH) manner. The conformations of arrayed OADs are synchronized in either microtubule‐binding state. The array involves extensive network interactions and is coordinately remodeled when OADs take one step forward from MTBS‐1 to MTBS‐2. The TTH interactions remain nearly unchanged in both states but need to be broken to allow MTBS alteration during mechanochemical cycle. Nucleotide treatment on the OAD‐MTD array reveals that it is the ATP hydrolysis that temporarily relaxes the TTH interfaces to allow free nucleotide cycle of downstream OADs. In combination with previously reported cryo‐ET structures, we propose a most detailed mechanism of how OADs coordinate with each other to move one step on MTD and a possible model for ciliary beat propagation.