- Intrinsically disordered proteins (IDP) are a class of proteins that lack a three-dimensional structure and their prevalence and diverse functions in the cell have only been discovered relatively recently. The intermediate chain (IC) subunit of the microtubule motor protein complex dynein contains an N-terminal disordered region, N-IC, which is central to both dynein assembly and regulation. The disordered N-IC contains binding sites for dynein light chain subunits, and dynein regulatory proteins---dynactin and NudE/Nudel, whose binding sites on IC are overlapping and interactions are mutually exclusive. Light chains binding and IC phosphorylation are suggested to affect dynein-regulator interactions, yet the mechanism and evolution of dynein regulation by dynactin and NudE/Nudel are unknown. This dissertation is aimed at characterizing the interactions between N-IC, light chains, and regulatory proteins in different species to gain insights into how the interplay of protein disorder, protein phosphorylation, and alternative splicing regulates protein-protein interactions using thermodynamical and structural biophysics methods.
Chapter 1 gives an introduction to IDPs including their biophysical properties, the basis for their diverse functions, general mechanisms for interactions, and methods to study IDPs with the emphasis on NMR spectroscopy. Chapter 2 provides background on the dynein complex including structural and assembly properties, subunit composition. Background information on the two dynein regulators of interest---dynactin and NudE/Nudel are also presented.
Chapter 3 presents studies on the structure and dynamics of yeast dynein N-IC (Pac11) and its interactions with light chain Dyn2 and dynactin subunit Nip100. We show that the N-Pac11 is primarily disordered except for an N-terminal single alpha helix (SAH) and another shorter nascent helix (LH) in the linker that separates the two Dyn2 binding sites. Both helices (SAH and LH) are required for Nip100 binding. Dyn2 binding induces structural changes in LH and alters the conformational ensemble of disordered Pac11 so that it promotes allosteric regulation of Nip100 binding. These results elucidate the role of light chains in dynein-dynactin interactions in yeast.
Chapter 4 presents studies on the structure and dynamics of mammalian (rat) dynein N-IC and the role of a single phosphorylation on different IC isoforms in dynactin subunit p150[superscript Glued] and NudE/Nudel binding to IC. We show that rat N-IC also contains a SAH domain and a second shorter helix but with much stronger helicity comparing to Pac11. The SAH domain is necessary and sufficient for both p150Glued and NudE/Nudel binding, although not optimal for p150[superscript Glued]. The phosphomimetic S84D mutation, which is not required in either binding event, decreases the affinity with p150Glued but not NudE/Nudel. Furthermore, we show that the p150[superscript Glued]-unfavorable conformation induced by S84D requires optimal coordination between the amino acid composition and length of disordered linkers, which contributes to the specificity of phosphorylation effect on IC isoforms. Together, these results reveal a multilayered dynein regulation in mammalian species.
Chapter 5 presents studies on the evolution of differential regulation of IC by p150[superscript Glued] and NudE/Nudel among three species. We show that folding coupled to binding and long range effects on IC are hallmarks of p150[superscript Glued] binding; p150[superscript Glued] and NudE/Nudel bind to non-coinciding surfaces on the SAH domain of N-IC; the differential dynein regulation mechanisms across species involve the second helix on IC; phosphorylation and alternative splicing are evolved in mammals as factors controlling the fine-tuning of dynein-regulator interactions.
Chapter 6 presents a summary of main results and conclusions in this dissertation and proposed future work to complement our on-going efforts to elucidate the mechanism and structural basis of dynein regulation. The work presented here characterizes structural and dynamic of the intrinsically disordered region of dynein intermediate chain (N-IC) in detail, and its interactions with two regulatory proteins. Together, the results provide answers to long-standing questions in the dynein field, such as what’s the effect of IC phosphorylation on dynein regulation, and how does IC bind to p150Glued and NudE/Nudel, and the first systematic studies on the evolution of dynein-regulator interactions.