Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains

Abstract

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...

Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein

“…Previous studies led us to hypothesize that the nucleotide state at AAA3 might play a role in the regulation of dynein by Lis1 (DeWitt et al, 2014; Nicholas et al, 2015; Toropova et al, 2014). To test this, we used a minimal S. cerevisiae dynein construct dimerized by GST (Dyn wt ) (Figure 1A) that is similar to full-length dynein in its motile properties (DeWitt et al, 2012; Reck-Peterson et al, 2006) and regulation by Lis1 (Huang et al, 2012).…”

“…For example, NudE/NudEL binds both dynein and Lis1 and has been implicated in many Lis1-dependent functions (Cianfrocco et al, 2015). Finally, an intriguing possibility is that a backward force exerted on dynein (such as that from a pulled cargo) could influence the nucleotide state of AAA3 (Nicholas et al, 2015). If a backward force on dynein promoted an apo state in AAA3, this would favor a single Lis1 β-propeller binding (at Site Ring ) and maintain dynein in a high MT affinity state, which would be advantageous when dynein is transporting a high load cargo.…”

“…The nucleotide state at AAA3 regulates dynein’s motility by acting on its mechanochemical cycle (DeWitt et al, 2014; Nicholas et al, 2015): stepping dynein contains ADP at AAA3, while ATP or no nucleotide (“apo”) at this site results in a slower dynein that is tightly bound to MTs (DeWitt et al, 2014). This AAA3-mediated inhibition occurs even when ATP binds to AAA1, which normally triggers MT release.…”

Chemomechanical regulation of myosin Ic cross-bridges: Deducing the elastic properties of an ensemble from single-molecule mechanisms

“…The bond interface consists of multiple partial charges that lead to complex unbinding pathways through multiple states in the free-energy landscape [71–74]. To capture the characteristic behavior, we use the force factor of Eq 17 that has been used previously to describe the chemomechanical cycle of kinesin-1 and myosin V [75–79].…”

Molecular machines

“…Ryota Iino is currently analysing the mechanical cycle of chitinase, a processive molecular machine that moves unidirectionally along crystalline chitin. Finally, Arne Gennerich is doing some of the best current work on the stepping mechanism of cytoplasmic dynein, which hauls cargo rapidly towards the minus ends of microtubules in cells (Nicholas et al 2015).…”