Synthetic cargo adaptors reveal molecular features that can enhance dynein activation.

Cytoplasmic dynein-1 (dynein) is the primary retrograde-directed microtubule motor in most eukaryotes. To be active, dynein must bind to the dynactin complex and a cargo-specific adaptor to form the . There are nearly 20 adaptors that, despite having low sequence identity, all contain two discrete domains that mediate binding to the same regions of dynein and dynactin. Additionally, all adaptors seem to generate active transport complexes with grossly similar structures. Despite these similarities, active transport complexes formed with different adaptors show differences in their velocity, run length, and microtubule binding affinity. The molecular features in adaptors that underlie the differences in activity is unknown. To address this question, we first generated a library of synthetic adaptors by deleting or systematically swapping characterized dynein and dynactin binding domains for four endogenous, model adaptors, NINL, BicD2, KASH5, and Hook3. We then used binding assays and TIRF-based motility assays to assess each synthetic adaptors' ability to bind and activate dynein and dynactin. First, we found that the adaptors' coiled-coil domains, which bind dynactin and the tail domain of dynein, are necessary and sufficient for dynein activation. Second, we found that all endogenous adaptors could be modified to yield a synthetic adaptor that formed more motile active transport complexes, which suggests that there is no selective pressure for adaptors to maximize dynein motility. Indeed, our data suggest that some endogenous adaptor sequences may have evolved to generate active transport complexes that are only moderately motile. Finally, we found that one synthetic adaptor was hyperactive and generated active transport complexes that moved faster, farther, and more frequently than all other endogenous and synthetic adaptors. By performing structure-function analyses with the hyperactive adaptor, we discovered that increased random coil at key positions in an adaptor sequence increases the likelihood that dynein-dynactin-adaptor complexes that assemble will be motile. Our work supports a model where increased adaptor flexibility facilitates a type of kinetic proofreading that specifically destabilizes improperly assembled and inactive dynein-dynactin-adaptor complexes. These results provide insight into how differences in adaptor sequences could contribute to differential dynein regulation.