Structural Ensembles of Intrinsically Disordered Proteins Depend Strongly on Force Field: A Comparison to Experiment

Abstract

Cytoplasmic dynein is a protein complex responsible for transporting cellular cargos on microtubules. Dynein's activities are regulated by other proteins including dynactin which mediates dynein-cargo interactions and increases dynein's processivity. The intermediate chain (IC) of dynein binds to dynactin p150Glued subunit, an interaction central to dynein regulation. The N-terminal domain of IC, N-IC is partially disordered, and contains binding site for a coiled-coil domain of p150Glued and a serine-rich region known to undergo phosphorylation in vivo. There are conflicting results to the effect of phosphorylation on dynactin binding. Using different techniques, one group showed phosphorylated S84 and a phosphomimetic mutant S84D abolished p150Glued binding in vitro while another group demonstrated S84D was still able to bind. We use mutagenesis, constructs of varying length, isothermal titration calorimetry (ITC) and NMR spectroscopy to identify the effect of phosphomimetic S84D on structure of IC and its ability to bind p150Glued. Backbone assignments of IC2C1-96 and secondary chemical shifts show residues 4-38 (helix 1) and 52-66 (helix 2) adopt helical structures and the rest of the protein is disordered. NMR and ITC titration studies identify the first 44 residues as sufficient for binding. The phosphomimetic mutant IC2CS84D abolishes p150Glued binding, without causing major changes in structure and dynamics as detected from NMR measurements. Chemical shift comparison between WT and S84D shows long-range effects on residues in the linker between helix 1 and 2, suggesting conformational changes in the linker. Mutations in the linker region restore p150Glued binding. Together, these results demonstrate for the first time phosphorylation in a disordered region affects binding at a distant site in the sequence, and is fine-tuned by changes in a disordered linker separating two structured domains. This regulatory control of dynein-dynactin interactions is key to cargo-and temporal-specific alterations in dynein transport.

Comparison of force fields for Alzheimer's A: A case study for intrinsically disordered proteins

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The prediction of topologically partitioned intra-atomic and inter-atomic energies by the machine learning method kriging

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