Abstract

Microtubule-associated proteins (MAPs) perform diverse functions in cells. These functions are dependent on their interactions with microtubules. Dynactin, a cofactor of dynein motor, assists the binding of dynein to various organelles and is crucial to the long-distance processivity of dynein-based complexes. The largest subunit of dynactin, the p150glued, contains a N-terminus segment that is responsible for the microtubule-binding interactions and long-range processivity of dynactin. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150glued N-terminal region, both free and in complex with polymerized microtubules. This 191-residue region encompasses the CAP-Gly domain, the basic domain and serine-proline-rich (SP-rich) domain. We demonstrate that the basic and SP-rich domains are intrinsically disordered in solution and significantly enhance the binding affinity to microtubules as these regions contain the second microtubule-binding site on the p150Glued subunit. The majority of the basic and SP-rich domains are predicted to be random-coil, while the segments S111-I116, A124-R132 and K144-T146 in the basic domain contain short -helical or {beta}-sheet structures. These three segments possibly encompass the microtubule binding site. Surprisingly, the protein retains high degree of flexibility upon binding to microtubules except for the regions that are directly involved in the binding interactions with microtubules. This conformational flexibility may be essential for the biological functions of the p150Glued subunit.\n\nSTATEMENT OF SIGNIFICANCEMicrotubule-associated proteins (MAPs) perform diverse functions in cells. Many of them comprises intrinsically disordered regions, whose structural flexibility are central to microtubule-based cellular functions of MAPs. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150glued N-terminal region encompassing the CAP-Gly domain, the basic domain and serine-proline-rich (SP-rich) domain, both free and in complex with polymerized microtubules. The results reveal that the basic and SP-rich domains are largely unstructured and retains high degree of flexibility upon binding to microtubules except for the regions that are possibly involved in the binding interactions with microtubules. This approach is informative for dynamics studies of intrinsically disordered MAPs and other disordered proteins in large biological assemblies.