Structural basis for affinity switching and biased movements of cytoplasmic dynein

Noritaka Nishida1,3, Yuta Komori2,3, Osamu Takarada1,3, Atsushi Watanabe1, Satoko Tamura1, Satoshi Kubo1, Ichio Shimada1*, Masahide Kikkawa2*
1 Graduate School of Pharmaceutical Sciences, 2 Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, 3 Equal contributions * Corresponding authors

Introduction
Figure 1 Figure 2
Cytoplasmic dynein is a motor protein that utilizes the energy of ATP to move along microtubules (MTs) toward the minus ends. The
movement of dynein on MT is achieved by alternate switching between weak and strong binding states of its microtubule binding domain
(MTBD). Affinity switching of the MTBD is thought to be achieved by nucleotide-state transition in the dynein ATPase cycle (Fig. 1).
Primary questions in this study are as follows.
1. Since the proposed model is mainly based on the “snapshots” of the motor domain in the different nucleotide states in the absence
of MTs, the temporal sequence of conformational changes within the large motor is speculative. It has been shown that ATP
hydrolysis allows the MTBD to return to a strong binding state[1], but it has also been proposed that MT binding of dynein promotes
ATP hydrolysis[2]. Therefore, it is unclear how the events in the ATPase domain (ATP hydrolysis and Pi release) is coupled with MT-
binding of the MTBD (corresponding to Fig. 1, state 2-4).
2. Secondly, the structural mechanism of the two-way communication between the ATPase domain and MTBD remains elusive.
Biochemical studies demonstrated that the distinctive changes in the affinity for MTs was caused by the registry exchange of the stalk
between α-registry (a high-affinity state) and +β-registry (a low-affinity state) which reflects the sliding of CC1 with respect to CC2 by
one-turn of α-helix (Fig. 2)[2][3]. However, the atomic details of the conformational difference of the MTBD in the high- and low-affinity
states remain elusive.
[4] Roberts, Anthony J., et al. "Functions and mechanics of dynein motor proteins.“ [5] Carter, Andrew P., et al. "Crystal structure of the dynein
3. Thirdly, it is unknown why the movement of dynein motor along microtubules is biased toward the minus end direction. Nature reviews Molecular cell biology 14.11 (2013): 713. motor domain." Science 331.6021 (2011): 1159-1165.

In this study, we exploited the disulfide cross-linking to regulate the MTBD either in the high or low-affinity states. We determined the NMR structures of the MTBD in the low and high-affinity states, and obtained
high resolution cryo-EM structure of MT-bound MTBD in the high-affinity and affinity-unlocked states. In addition, the difference in the interaction sites with MT between the strong and weak binding MTBD was
revealed by NMR. Those data clarified the structural changes of the MTBD, which explain the affinity regulation mechanism of cytoplasmic dynein, and provided the structural mechanisms for the directional
preference of dynein on the MT track.

Methods & Results

1. MT affinity of MTBD-WT, Low and High We performed NMR structural analysis for MTBD-WT, Low and High. The HSQC spectrum of MTBD-WT
We made the 137-residue construct encoding K3096-E3232 of cytoplasmic dynein of Saccharomyces implies that the MTBD without the S-S linkage is in an equilibrium between MTBD-Low and High (Fig.
cerevisiae. This construct, termed MTBD-WT was expressed by E. coli and purified to homogeneity. To 4A). Though the MTBD-Low structure was found to be in the +β-registry, the MTBD-High showed the
stabilize the MTBD in the low- and high-affinity state (MTBD-Low and MTBD-High), we employed a distinctive displacement of CC1 with respect to CC2 by a half-turn of α-helix, possibly reflecting the
disulfide locking strategy by introducing a pair of cysteine mutations on CC1 and CC2 (Fig. 3A). We then MTBD’s rigidity (Fig. 4B). We refer to the registry of MTBD-High as “semi-α”, since the sliding of the CC1
conducted MT co-sedimentation assay and calculated the dissociation constants (Kd) (Fig. 3B). is in the intermediate position of +β- and α-registry.
Figure 3 Furthermore, we conducted the TCS experiment, a nuclear magnetic resonance measurement technique
A B for the precise identification of the interfaces of protein-protein complexes[6]. It indicated that H3 plays
CC2 the most significant role for high affinity interaction of the MTBD (Fig. 4C)
C
3. Cryo-EM structure of MT-bound MTBD-High and affinity-unlocked MTBD
N
CC1
We then performed cryo-EM single particle analysis of the MTBD-High in complex with MTs (Fig. 5A).
CC2
We also analyzed the complex in the presence of DTT to cleave the disulfide bond (Fig. 5B). The final
C resolutions of DTT(-) and DTT(+) structure were 3.94 Å and 3.66 Å, respectively. We built atomic model
N
structures by a flexible fitting method using our NMR-derived MTBD-High structure as an initial model.
CC1 Interestingly, we found that the two MTBD-High/MT complexes +/- DTT were very similar to each other
(Fig. 5C), suggesting that the MT-bound MTBD-High structure was formed mainly by MT-binding, not by
the disulfide bond. In the following, we mainly use the atomic model of MTBD-High/MT-DTT(+)
2. NMR structural analysis of MTBD-WT, Low and High (referred to as MTBD-Bound).
Figure 4 C MTBD-Low Comparing the MTBD-Low, Figure 5
A B
A High and Bound, we found
MTBD-Bound (DTT(-)) ) MTBD-Bound (DTT(+))
that the CC1 of the MTBD-
Bound slided further toward
the N-terminal direction
compared to the MTBD-High
(corresponding to the semi-α-
MTBD-High registry), resulting in the
registry shift by one-turn of
α-helix from the MTBD-Low
(corresponding to +β-registry),
B i.e. turning into the α-registry. C D
Therefore, the stalk’s coiled-
coil shifting caused by ATP
hydrolysis may be insufficient
for stabilizing the MTBD in
the α-registry, and that the α-
registry is likely stabilized
upon MT binding by induced
fit mechanism.

Discussion
This study revealed three conformational states of the dynein MTBD
with a distinctive coiled-coil registry (+β, semi-α, and α, summarized
Figure 6 B
in Fig. 6A). Based on the conformational changes of the MTBD in the A
absence and presence of MTs in this study, we propose that the
mechanochemical cycle of dynein could proceed with two distinctive
pathways, namely “ATPase-driven pathway” and “MT-binding induced α β
pathway” (Fig. 6B). The latter pathway is based on a previous
biochemical study which showed the ATPase activity is enhanced in
the α-registry compared to the +β-registry[2].
Our results may provide a clue for the dynein’s biased movement
toward the minus end direction. The MTBD in the weak binding state
makes initial attachment to MTs via H1 and H6, while the interaction
mediated by H3 has not formed yet (Fig. 6C-1). When the force is
applied toward the backward direction, the stalk would tilt backward,
thereby pushing the H3 toward β-tubulin (Fig. 6C-2), which would be
preferable for forming high affinity binding (Fig. 6C-3). In the two- C
headed motor domain stepping on the MT track, the leading head in 1 2 3

the low affinity state searches on the MTs to form a new binding site,
while the lagging head in the high affinity state remains attached on
the MTs. The angle between stalk and MTs would become more acute
when the leading head steps forward, than when steps backward.
Therefore, for the same reason as the backward force is applied, the
forward movement would be favored over the backward. minus end

Conclusion
➢ We determined MT-unbound structures of the dynein MTBD both in the low- and high-affinity states
and investigated the difference in the residues involved in the interaction with MT using NMR technique.
➢ We obtained cryo-EM structures of the high-affinity and affinity-unlocked MTBD in complex with MTs.
➢ Based on the results, we hypothesized two distinctive pathways for dynein’s MT-binding.
➢Furthermore, we proposed a possible mechanism of the dynein’s biased movements on the MT tracks.
References
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