Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated

Abstract

SummaryCytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves poorly on its own or how it is activated by dynactin. Here, we present a cryoelectron microscopy structure of the complete 1.4-megadalton human dynein-1 complex in an inhibited state known as the phi-particle. We reveal the 3D structure of the cargo binding dynein tail and show how self-dimerization of the motor domains locks them in a conformation with low microtubule affinity. Disrupting motor dimerization with structure-based mutagenesis drives dynein-1 into an open form with higher affinity for both microtubules and dynactin. We find the open form is also inhibited for movement and that dynactin relieves this by reorienting the motor domains to interact correctly with microtubules. Our model explains how dynactin binding to the dynein-1 tail directly stimulates its motor activity.

Molecular basis for dyneinopathies reveals insight into dynein regulation and dysfunction

“…In the cases of R241L and N283R, the gain-offunctions observed in vitro are possibly causative of the in vivo deficiencies, indicating that a faster or more processive motor is not advantageous for the spindle positioning function (see Discussion regarding K1475Q). This is consistent with recent studies that showed gain-of-functions in dynein (or dynein regulators) can lead to defects in dyneinmediated neuronal maturation 85 , or spindle orientation 72 .…”

“…5D) -which would result in fewer motor complexes being engaged for a spindle movement event -is the basis for cellular dysfunction. Given the auto-inhibited Phi particle exhibits reduced affinity for dynactin 72 , the altered localization pattern of the mutant may be due to disruption of a similar autoinhibitory conformation in yeast, consequent increased dynactin binding, and thus an increase in the frequency of cortical off-loading events 63 . Previous studies have suggested that dynein's interaction with dynactin is a limiting step in the delivery of dynein-dynactin complexes to cortical Num1 sites 77 .…”

SmartBac, a new baculovirus system for large protein complex production

“…4j). These include the human COPI complex (7 subunits, 558 kDa) (Wang et al, 2016), cytoplasmic Dynein complex (12 subunits, 1380 kDa) (Zhang et al, 2017), CSN complex (8 subunits, 343 kDa) (Mosadeghi et al, 2016) and SCF complex (5 subunits, 180 kDa) (Zheng et al, 2002). The recombinant COPI complex sample has been used to study the structure of coatomer in its soluble form (Wang et al, 2016).…”

Turning dyneins off bends cilia

“…Structural studies of canonical cytoplasmic dynein and the dynein that powers retrograde intraflagellar transport revealed an auto‐inhibited state (the φ particle), in which two motor heads stack together forming an interface that buries the linkers that cross the AAA ring and in which the microtubule‐binding stalks are crossed, thus structurally inhibiting the main mechanical elements (Torisawa et al, ; Pigino & King, ; Toropova et al, ; Zhang et al, ). This structure is not observed in axonemal dyneins in situ, and thus the inhibited axonemal dynein state seen by Lin and Nicastro () has a distinct structural basis.…”