The AAA3 domain of cytoplasmic dynein acts as a switch to facilitate microtubule release

Abstract

Cytoplasmic dynein is an AAA+ motor responsible for intracellular cargo transport and force generation along microtubules (MTs). Unlike kinesin and myosin, dynein contains multiple ATPase subunits, with AAA1 serving as the primary catalytic site. ATPase activity at AAA3 is also essential for robust motility, but its role in dynein's mechanochemical cycle remains unclear. Here, we introduced transient pauses in Saccharomyces cerevisiae dynein motility by using a slowly hydrolyzing ATP analog. Analysis of pausing behavior revealed that AAA3 hydrolyzes nucleotide an order of magnitude more slowly than AAA1, and the two sites do not coordinate. ATPase mutations to AAA3 abolish the ability of dynein to modulate MT release. Nucleotide hydrolysis at AAA3 lifts this 'MT gate' to allow fast motility. These results suggest that AAA3 acts as a switch that repurposes cytoplasmic dynein for fast cargo transport and MT-anchoring tasks in cells.

Figure 1. ATPase mutations to the AAA3 site abolish nucleotide-dependent release of dynein from MTs

(a) The dynein motor domain consists of a ring of six AAA+ sites (numbered in black). ATP hydrolysis at AAA1 and AAA3 are required for robust motility. (b) Images of MTs decorated with TMR-labeled dynein monomers were recorded before and after flow with the indicated solution. Scale bar is 2 µm. (c) The fraction of monomers released for different nucleotide treatments. Error bars represent s.e.m. of 15 MTs for each condition. (d) Force-dependent release rates of AAA1_E–Q and AAA3_E–Q mutants. MT release rates of the WT monomer in the presence and absence of ATP are shown for comparison. Error bars on each bin represent s.e.m. of 100 release events. (e) The average release rates of dynein mutants under 1–3 pN of load either toward the minus- or plus-end of MTs (n = 192 for the plus-end direction and 164 for the minus-end direction, ±95% confidence interval).

Figure 2. Dynein motility is gated by MT release in the absence of an active AAA3 site

(a) Schematic of Dyn1_331kD dimerized through GST. (b,c) Kymographs of TMR-labeled WT (b) and AAA3_K–A (c) walking along axonemes at 1 mM ATP. (d,e) Single-molecule velocities of WT (d) and AAA3_K–A (e) at 1 mM ATP and 0–100 mM KCl. Error bars represent s.e.m. (n >88 for AAA3_K–A, n >126 for WT per concentration). (f,g) MT-stimulated ATPase activity of WT (f) and AAA3_K–A (g) as a function of added KCl. Error bars represent s.e.m of 3 technical replicates.

Figure 3. Single-molecule enzyme inhibition of dynein by ATPγS

(a) AAA3 may facilitate MT release either by directly coordinating its ATP hydrolysis cycle with that of AAA1 (left), or by controlling an allosteric “switch” that allows AAA1 to communicate with the MTBD (right). (b) Representative traces of WT dynein motility at 1 mM ATP and varying amounts of ATPγS (n = 120). The frequency of pauses increases at higher ATPγS concentrations. (c) The residence time histogram of a single motor for each 50 nm distance travelled along its path. Peaks indicate locations in which the motor experiences a prolonged pause. (d) The cumulative residence time histogram of 120 traces fits well to biexponential decay (red curve). The areas under the red and yellow curves represent the slow and fast populations, respectively. (e) PD (blue circles) as a function of ATPγS concentration. Red curves represents a fit to to the Hill equation. (f) Velocity of WT dynein as a function of ATP concentration (black circles) fits well (R² = 0.996) to Michaelis-Menten kinetics (blue curve) and deviates away (R² = 0.72) from Hill Equation with n = 2 (red dashed curve). Error bars represent s.d. of 120–246 traces of single motors.

Figure 4. Motility of the AAA3_K–A mutant is insensitive to ATPγS

(a) Representative traces of QD-labeled AAA3_K–A motility at 1 mM ATP and 0 – 1,000 µM ATPγS (n = 100). Long pauses are not visible even at 1,000 µM ATPγS. (b) The velocity of AAA3_K–A as a function of ATPγS concentration. Error bars represent s.e.m of 87–161 traces. (c) PD of AAA3_K–A remains unaffected by ATPγS concentration. Error bars represent s.e.m of PDs calculated from 87–161 traces. (d,e) ATPase rates of WT (d) and AAA3_K–A (e) at saturating MT concentrations. Colors represent different concentrations of ATPγS. All plotted data are from 3 technical replicates. (f) ATPγS hydrolysis rates of WT and AAA3_K–A at saturating MTs in the absence of ATP. ATPγS has a weak affinity (K_M(ATPγS) = 500 ±110 µM for WT and 425 ± 98 µM for AAA3_K–A, mean ± 95% c.i.), and a relatively fast turnover rate (k_cat(ATPγS) = 4.52 ± 0.42 s⁻¹ for WT and 2.34 ± 0.19 s⁻¹ for AAA3_K–A, mean ± 95% c.i.), consistent with poor inhibition of AAA1.

Figure 5. A minimal dynein construct with one AAA+ ring shows two-state pausing behavior

(a) Schematic (inset) representing the minimally processive SRS-WT heterodimer. Representative traces of QD-labeled SRS-WT in the presence of 1 mM ATP, and 0, 10, or 100 µM ATPγS show that increasing the ATPγS concentration results in more frequent pauses and slower motility (n = 259). (b) Normalized histograms of the velocities of SRS-WT over 1 second intervals at varying concentrations of ATPγS. These histograms intersect at 17 nm s⁻¹ (blue arrows), consistent with a two-state transition between “fast” and “slow” states. The inset, which shows each histogram with the 0 µM ATPγS histogram subtracted, reveals the velocity distributions of the two populations. (c) PD of SRS-WT at different ATPγS concentrations. Gray curve represents the fit of the data to the Hill equation with n fixed at 1.0. PD_MAX (red dotted line) is 0.0063 ± 0.0004 pauses nm⁻¹ (mean ± 95% c.i.). (d) Pause duration at different ATPγS concentrations. Error bars represent s.e.m. of 81–277 pauses calculated from 73–259 traces.

Figure 6. The SRS-WT heterodimer takes long runs between pauses at equimolar concentrations of ATP and ATPγS

(a,b,c) Representative traces of SRS-WT motility at 10:1 (a), 2:1 (b) and 1:1 (c) ATP:ATPγS ratios, respectively (n = 64, 99, and 117 traces for 100 µM, 200 µM, and 1 mM ATP, respectively). Long runs of fast motility (>15 nm s⁻¹) are shown in red. (d,e,f) Velocity (d), the frequency of fast runs (e) and lengths of fast runs (f) of SRS-WT motility at different ratios of ATP to ATPγS. Error bars represent s.e.m. of 64, 99, and 117 traces for 100 µM, 200 µM, and 1 mM ATP, respectively.

Figure 7

ATP hydrolysis cycle at AAA1 drives the stepping motility of dynein. (Left) When AAA3 is in the apo state, communication between AAA1 and the MTBD is blocked, resulting in slow, force-dependent MT release, and consequent slow progression through the AAA1 hydrolysis cycle (τ_cycle = 1.2 s)^,. (Right) When ATP binds to AAA3 and is hydrolyzed, the allosteric circuit connecting AAA1 and the MTBD is completed. In this state, ATP binding at AAA1 leads to fast release from the MT, and subsequent progression through the AAA1 hydrolysis cycle (τ_cycle = 0.13 s). Cycle times are calculated from the bulk ATPase rates per head. This AAA3 controlled switch may play an essential role in repurposing of dynein for intracellular transport (i.e. fast MT release) and anchoring (i.e. slow MT release) functions.