Interplay of Disorder and Sequence Specificity in the Formation of Stable Dynein-Dynactin Complexes

Abstract

Cytoplasmic dynein is a eukaryotic motor protein complex that, along with its regulatory protein dynactin, is essential to the transport of organelles within cells. The interaction of dynein with dynactin is regulated by binding between the intermediate chain (IC) subunit of dynein and the p150 Glued subunit of dynactin. Even though in the rat versions of these proteins this interaction primarily involves the single a-helix region at the N-terminus of the IC, in Drosophila and yeast ICs the removal of a nascent helix (H2) downstream of the single a-helix considerably diminishes IC-p150 Glued complex stability. We find that for ICs from various species, there is a correlation between disorder in H2 and its contribution to binding affinity, and that sequence variations in H2 that do not change the level of disorder show similar binding behavior. Analysis of the structure and interactions of the IC from Chaetomium thermophilum demonstrates that the H2 region of C. thermophilum IC has a low helical propensity and establishes that H2 binds directly to the coiled-coil 1B (CC1B) domain of p150 Glued , thus explaining why H2 is necessary for tight binding. Isothermal titration calorimetry, circular dichroism, and NMR studies of smaller CC1B constructs localize the region of CC1B most essential for a tight interaction with IC. These results suggest that it is the level of disorder in H2 of IC along with its charge, rather than sequence specificity, that underlie its importance in initiating tight IC-p150 Glued complex formation. We speculate that the nascent H2 helix may provide conformational flexibility to initiate binding, whereas those species that have a fully folded H2 have co-opted an alternative mechanism for promoting p150 Glued binding.

Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function

“…A compound binding with a high affinity may freeze the inherent internal flexibility of NUPR1, impacting its activity in other ways. It is worth to note that a residual flexibility has been shown to be necessary to carry out the function of a number of proteins: for instance, neurotensin needs to maintain its flexibility around the crucial residue Tyr11 when the protein is bound to neurotensin receptor 1 [47], and the same applies for a whole nascent helix of dynein when it is bound to dynactin [48]. As a second possible explanation for the biological effects we observed, it has been recently argued that an effective strategy for drug discovery in IDPs relies in exploiting the entropic contributions due to the protein chain flexibility, which would increase the affinity towards ligands by shifting the population of disordered conformations [49,50].…”

Consensus nomenclature for dyneins and associated assembly factors

“…The intermediate chains of metazoan dynein 1 connect it to another multi-subunit complex known as dynactin ( Loening et al, 2020 ). Dynactin is built around a filament of the protein encoded by ACTR1A (actin-related protein 1A).…”

“…A single adaptor protein sandwiches between dynactin and dynein, where it interacts with the dynein heavy chain tails and the light intermediate chain and along the length of the dynactin complex ( Gonçalves et al, 2019 ; Reck-Peterson et al, 2018 ). There are currently ≥12 known cargo adaptor proteins, which are encoded by HOOK1 , HOOK2 , HOOK3 , BICD2 , BICDL1 , BICDL2 , RAB11FIP3 , RASEF , CRACR2A , NIN , NINL , and SPDL1 ( Barisic et al, 2010 ; Casenghi et al, 2005 ; Dona et al, 2015 ; Gonçalves et al, 2019 ; Horgan et al, 2010 ; Lee et al, 2018 ; Loening et al, 2020 ; Olenick et al, 2016 ; Vallee et al, 2021 ; Wang et al, 2019 ). Other protein cofactors may also be required for dynein recruitment to their cargoes.…”

Cargo-Mediated Activation of Cytoplasmic Dynein in vivo

“…Following the MT-binding domain are the coiled-coil domains CC1 and CC2, and CC1 interacts with the N-terminus of dynein IC in biochemical assays (Karki and Holzbaur, 1995;Vaughan and Vallee, 1995;King et al, 2003). The CC1 domain of p150 can be further divided into CC1A and CC1B ( Figures 1B,C), and CC1B contains a dynein-IC-binding domain (McKenney et al, 2011;Loening et al, 2020) whose function may be modulated by the binding of CC1A (Tripathy et al, 2014;Saito et al, 2020). Two other subunit of the dynactin complex, p22/p24 and p50 dynamitin that forms an oligomer (Echeverri et al, 1996;Karki et al, 1998;Melkonian et al, 2007), together with part of the p150 subunit, form a "shoulder/side arm" adjacent to the Arp1 mini-filament, and the p50 oligomer was proposed to function as a template for Arp1 mini-filament assembly (Urnavicius et al, 2015).…”