Protein Disorder in Dynein Regulation by Dynactin and NudE

Abstract

Cytoplasmic dynein is a multi-subunit protein complex responsible for retrograde transport of diverse cellular cargoes along microtubules. Dynein is comprised of heavy chains responsible for motor activity, and intermediate chain (IC) and light chains for cargo attachment and regulation. Dynein light chain LC8 is conserved across species and its binding promotes dimerization and stabilization of IC but its effect on dynein regulation remains unclear. Dynein activity is regulated by other proteins such as dynactin, which is essential for most dynein activities; and NudE, which functions in targeting dynein to the kinetochores. LC8, dynactin subunit p150Glued and NudE coiled-coil domain all bind the disordered N-terminal domain of IC. Using NMR and ITC on Saccharomyces cerevisiae and Drosophila melanogaster constructs, we show that the N-terminal helix of IC is a single α-helix (SAH) instead of a dimeric coiled-coil as predicted. NMR titrations map the exact residues of IC involved in binding to both p150Glued and NudE and the accompanying changes in structure and dynamics, while ITC experiments identify the domains of IC necessary for full binding affinity. In yeast, p150 and NudE both bind the IC SAH domain, and both interactions are enhanced when LC8 is present. In Drosophila, p150 and NudE both bind to the SAH domain but p150 also binds to a second site on IC, a nascent helix separated from the SAH domain by a 4-residue linker. A dimeric IC formed by cross-linking IC chains has increased affinity to p150 but similar affinity to NudE, indicating that bivalency causes differential effects on binding regulatory proteins, and illustrating intriguing species variation in dynactin binding to IC. These studies illustrate the importance of multiple techniques to elucidate interplay of order and disorder in providing both structural and functional versatility for complicated systems.