Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.cytoplasmic dynein | mechanosensing | optical tweezers | AAA+ ATPases | microtubules N umerous eukaryotic cellular processes require motion and force generated by cytoskeletal motor proteins, among which cytoplasmic dynein (hereinafter, "dynein") is unique for its size, complexity, and versatility. As a homodimeric, divergent AAA+ ATPase (AAA: ATPase associated with various cellular activities), dynein drives the majority of microtubule (MT) minusend-directed motility in most eukaryotes (1). The motor functions as a massive protein complex (2), but its catalytic core consists of two identical heavy chains, each with six AAA modules (AAA1-6) linked in tandem to form a ring (Fig. 1A). AAA1-4 bind nucleotides, whereas AAA5 and -6 are structural (3, 4). A ∼15-nm "stalk" emerging from AAA4 (3, 4) separates the AAA modules from the MT-binding domain (MTBD). The stalk configuration influences both MT affinity and ATPase activity (5) and thereby mediates bidirectional allosteric communication between the AAA ring and the MTBD (3, 6). Finally, a ∼10-nm "linker" also emerges from the ring and undergoes cyclic reorientations that generate force and displacement (7-9).For dynein to "walk," one motor domain ("head") must remain MT-bound while the other moves (10-13), thus requiring coordination of the "internal" cycles of both heads. Dynein may use allosteric mechanosensing (possibly through the stalk) to differentiate be...

Cytoplasmic dynein is the most complex cytoskeletal motor protein and is responsible for numerous biological functions. Essential to dynein’s function is its capacity to respond anisotropically to tension, so that its microtubule-binding domains bind microtubules more strongly when under backward load than forward load. The structural mechanisms by which dynein senses directional tension, however, are unknown. Using a combination of optical tweezers, mutagenesis, and chemical cross-linking, we show that three structural elements protruding from the motor domain—the linker, buttress, and stalk—together regulate directional tension-sensing. We demonstrate that dynein’s anisotropic response to directional tension is mediated by sliding of the coiled-coils of the stalk, and that coordinated conformational changes of dynein’s linker and buttress control this process. We also demonstrate that the stalk coiled-coils assume a previously undescribed registry during dynein’s stepping cycle. We propose a revised model of dynein’s mechanochemical cycle which accounts for our findings.

Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a ‘cap’ over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function.

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