Multiple essential processes, including those associated with signaling, cell division, and viral replication are regulated by post-translational modifications of proteins that will either promote or inhibit complex assembly. The molecular bases for these effects, especially those involving autoinhibition aided by intrinsically disordered proteins (IDPs), are poorly understood. Here we elucidate the structural basis of autoinhibition and the role of intrinsic disorder, phosphorylation, and linker length in the regulation of large macromolecular complexes.
The dynein complex, a 1.6-MDa motor protein complex uses the energy of ATP hydrolysis to translocate along microtubules and carry cellular organelles from one subcellular locale to another. Dynein malfunction in the Golgi apparatus for example is an early feature in neurodegenerative disorders such as ALS and AD. Central to dynein multifunctions is its interaction with dynactin p150Glued through the intrinsically disordered dynein intermediate chain IC. IC also binds the nuclear distribution protein (NudE) at an overlapping site with p150Glued, and three dimeric dynein light chains at multivalent sites. The disorder in IC has hindered cryo-electron microscopy and X-ray crystallography resolution of its structure and interactions. Using proteins from Chaetomium thermophilum (CT), a tractable model for IC interactions, we identify long range intramolecular interactions between the N-terminal single α-helix of IC which is the binding region for p150Glued and NudE, and an alpha helix corresponding to LC7 binding site, closer to the C-terminal of the disordered domain, thus causing autoinhibition or no binding to either p150Glued and NudE. We demonstrate that this autoinhibitions is relieved by assembly with the light chains or by phosphorylation.
Common features elucidated on this system can be extended to other dynamic complex assemblies that require binding to other proteins for their activation. One such system is the nucleocapsid protein N from the SARS-CoV2. We demonstrate how the nucleocapsid functions are regulated by specific phosphorylation in the intrinsically disordered SR region that links the RNA binding domain to the dimerization domain. The work underscores the importance of combining several biophysical techniques in addition to NMR to study non-structured proteins, offering a blueprint that is relevant for a wide range of systems.