Jao Lab
Dynein-cargo interactions
Cytoplasmic dynein 1 (dynein) is the motor protein complex that transports most cellular cargoes along microtubules toward the center of an animal cell. To become an active motor transporting cargoes, dynein binds its general cofactor dynactin and an activating cargo adaptor attached to a specific cargo (e.g., organelles, mRNAs).
Activating cargo adaptors typically contain long coiled-coils with specific motifs for binding dynein-dynactin. Coiled-coils, the protein structures formed by intertwining of ⍺-helices, are found in proteins throughout all three kingdoms of life. Despite its simplicity at the sequence architecture, it is one of the most versatile protein folds in nature, enabling diverse biological functions, ranging from providing structural stability of cellular structures, mediating signal transduction, to forming key components underlying molecular motor proteins.
A common theme has emerged that coiled-coils with divergent sequences can impart similar functions. For example, centrosomal proteins (60% are coiled-coils) across metazoans all nucleate microtubules, but they show limited sequence similarity. The same is true for ~20 known dynein cargo adaptors that bind and activate dynein using long coiled-coils with limited sequence conservation.
Using a combination of approaches in computational molecular biology and experimental cell biology, we aim to determine the molecular grammar that transforms the coiled-coils with diverse sequences to perform a unified function such as assembling the centrosome and mediating dynein-cargo interactions.
PCNT engages with dynein-dynactin using adaptor-like sequences
Dynein-mediated co-translational transport of PCNT facilitates centrosome maturation and mitotic spindle formation. How dynein engages and transports PCNT, however, remains unclear. We found that PCNT contains adaptor-like sequences that bind and activate dynein during its transport to the centrosome. Specifically, PCNT interacts with the dynein heavy chain (DHC) through a patch of acidic residues. This binding interface—involving the conserved tyrosine 827 and arginine 759 residues of DHC and two HBS1-like motifs of PCNT (N-HBS1-like and C-HBS1-like)—resembles the one between DHC and a canonical activating adaptor. Interestingly, PCNT appears to use two HBS1-like motifs to engage with DHC, similar to how the canonical HOOK3 adaptor binds DHC. HOOK3 is unique among activating adaptors that it uses two rather than one HBS1 motif to bind DHC.
Based on this new finding, we modified the model of co-translational transport of PCNT toward the centrosome, in which nascent PCNT polypeptides co-translationally assemble into an adaptor-like coiled-coil to engage and activate the retrograde transport of PCNT polysomes. This finding may represent a potential new mechanism of how dynein engages with its other cargoes, the one through a direct interaction without a cargo adaptor.
Bulk molecular features shape the coiled-coils to perform similar functions
In collaboration with Alan Moses's group, we found that bulk molecular features are under selective pressure to preserve functionality of a protein despite many changes at the amino acid sequences over time.
One example is the dynein-binding coiled-coils of 15+ dynein cargo adaptors and the PCNT residues 1393–1525, the coiled-coil-rich region responsible for dynein binding. These coiled-coils have little to no sequence similarity, but share a similar set of molecular features, including high in glutamate and leucine, but low in proline residues (see the heat map; the adaptor dynein-binding coiled-coils and PCNT residues 1393–1525 are clustered together). This similar set of molecular features, collectively termed an evolutionary signature, is apparently preserved to enable those coiled-coils to exert a common function of binding to dynein-dynactin.
We hypothesize that the bulk sequence features that alter coiled-coil properties represent an unappreciated means by which diverse structural folds and properties of coiled-coils are generated to perform various functions in the cell. We are testing this hypothesis by designing synthetic sequences based on specific evolutionary signatures and testing whether the resulting de novo designed coiled-coils can perform specific biological functions such as mediating dynein binding and transport.
