Polybivalency and disordered proteins in ordering macromolecular assemblies

Abstract

Intrinsically disordered proteins (IDPs) are prevalent in macromolecular assemblies and are thought to mediate protein recognition in complex regulatory processes and signaling pathways. The formation of a polybivalent scaffold is a key process by which IDPs drive early steps in macromolecular assemblies. Three intrinsically disordered proteins, IC, Swallow and Nup159, are core components, respectively, of cytoplasmic dynein, bicoid mRNA localization apparatus, and nuclear pore complexes. In all three systems, the hub protein LC8 recognizes on the IDP, short linear motifs that are fully disordered in the apo form, but adopt a β-strand when bound to LC8. The IDP/LC8 complex forms a bivalent scaffold primed to bind additional bivalent ligands. Scaffold formation also promotes self-association and/or higher order organization of the IDP components at a site distant from LC8 binding. Rigorous thermodynamic analyses imply that association of additional bivalent ligands is driven by entropic effects where the first binding event is weak but subsequent binding of additional ligands occurs with higher affinity. Here, we review specific examples of macromolecular assemblies in which polybivalency of aligned IDP duplexes not only enhances binding affinity and results in formation of a stable complex but also compensates unfavorable steric and enthalpic interactions. We propose that polybivalent scaffold assembly involving IDPs and LC8-like proteins is a general process in the cell biology of a class of multi-protein structures that are stable yet fine-tuned for diverse cellular requirements.

Keywords: Bivalency; Enthalpy–entropy compensation; Intrinsically disordered proteins; LC8; Macromolecular assembly; Poly-bivalent scaffold.

Figure 1

Scheme illustrating bivalent interactions in disordered proteins. A high entropic penalty is associated with monovalent interactions. Aligning two chains of a disordered protein by an artificial linker, black line (A), through inter-chain (self-association) interactions, red bars (B), or by their interactions with a dimeric protein, yellow triangles (C) reduces the entropic penalty of a second binding event, green spheres.

Figure 2

Thermodynamics of LC8 binding to monovalent and bivalent IC. (A) Representative ITC thermograms (top panels) and binding isotherms (bottom panels) for titration of IC_TL (left) and IC_LL (right) with LC8. (B) Binding free energies ΔG° (kcal/mol), and ΔCp (kcal/mol/K) are given for each step. The difference in free energy and heat capacity changes between the second and first binding events of LC8 to IC_LL (right) are expressed as ΔΔG° and ΔΔCp, respectively, and are shown in the center. Binding parameters for the first IC_LL/LC8 binding event are assumed to be similar to those to IC_L (left). Binding parameters for the second IC_LL/LC8 binding event are inferred from average values determined from direct measurements. Figures adapted from [5].

Figure 3

Changes in thermodynamic parameters between first and second LC8 binding events (ΔΔG°, grey diamonds; ΔΔH°, black circles; and Δ-TΔS°, white squares). Differences in association parameters at different temperatures between LC8 binding to monovalent apo-IC_LL and LC8 binding to bivalent IC_LL/LC8 (left), and between LC8 binding to monovalent Swa_MONOMER and LC8 binding to bivalent Swa_DIMER (right). The absence of temperature dependence (ΔΔCp of zero) shows that binding enhancement (ΔΔG°) is not due to enthalpic change (ΔΔH° of zero) but due to entropic change (Δ-TΔS° of same value as ΔΔG°). Figures adapted from [5], and [8].

Figure 4

Dyn2-Nup159 binding parameters and ITC data. Binding thermograms, isotherms, and computed thermodynamic parameters show both the effect of bivalency on binding enhancement and compensation of unfavorable interactions. (A) Representative isothermal titration plots of Dyn2 with one, two and three recognition motifs, respectively, with the corresponding binding constant shown at the bottom of each plot. The interaction with one recognition motif is too weak for data fitting. (B) A hypothetical model of the bound complex composed of two disordered chains of Nup159 (black) and three Dyn2 dimers (green) with Nup159 contact residues adopting β-strand conformations (black arrows). (C) Representative isothermal titration plots of Dyn2 with three, four and five recognition motifs, respectively, with the corresponding binding constant shown at the bottom of each plot, along with the enthalpic and entropic contributions for each binding events. (D) A model of the wild type Nup 159 complex with 5 Dyn2 dimers. The ITC-measured differences in the contribution of the entropy term suggest more flexibility with three Dyn2 (B), but more order and rigidity with five Dyn2 (D). Figure is adapted from [9].

Figure 5

Residue-specific NMR titration of Nup159 NH peak intensity with added Dyn2. Relative NH peak intensity versus residue numbers of non-proline Nup159 NH peaks in NMR spectra of Dyn2-bound Nup159 in the sequence 1075–1178. Shown are Nup:Dyn2 molar ratios of 1:0.8 (top) and 1:6 (bottom). Peak intensities are relative to the intensity of the same peak in apo Nup159, which is taken as one. This figure is adapted from [9] which also reports results at a 1:2 ratio. The models on the left show two chains of Nup159 (red) with five binding sites for Dyn2 (green). In the top model, a single Dyn2 dimer, indicated at the bottom, is added to two molar equivalents of Nup monomer, and its effect is manifested throughout the Nup159 chain, as indicated regions of attenuated intensity all along the Nup chain. In the bottom model, Dyn2 dimers saturate the five Dyn2 binding sites. The observed loss of peak intensity arises from peak broadening due to exchange, on the NMR time scale, of an NH between different local environments (e.g., in bound and unbound conformations), and/or from an increased population within the ensemble of Nup conformations having significantly greater rotational correlation time [20]. In the latter case, the NH being measured might, for example, fractionally exist in several-to-many conformations such that the same NH when in disordered and locally flexible conformations gives a sharp ‘random coil’ signal, and when in more collapsed and/or aggregated conformations with a significantly longer tumbling time and faster relaxation time gives a broadened signal.

Figure 6

Examples of LC8 binding partners having multiple recognition motifs in intrinsically disordered regions. Sequence-based predictions of order, disorder, coiled-coil and LC8 binding motifs are shown for residues 1000 – 2500 of Bassoon, a protein involved in the organization of the cytomatrix at the nerve terminals active zone; the intermediate chain (IC) subunit of the cytoplasmic dynein molecular motor; yeast nucleoporin, Nup159; Chica, a spindle-associated adaptor protein; the flagellar radial spoke protein 3, RSP3, and the Drosophila homologue of ASCIZ (dASCIZ), a DNA damage response and transcription factor. In all these examples, the putative LC8 sites are located within segments with high sequence disorder. Putative motifs are based on the following references: bassoon [21], Nup159 [9], IC[5], Chica[22], RSP3[23] and dASCIZ[24].