Multivalency in the assembly of intrinsically disordered Dynein intermediate chain

Abstract

Dynein light chains are thought to increase binding efficiency of dynein intermediate chain to both dynein heavy chain and dynactin, but their exact role is not clear. Isothermal titration calorimetry and x-ray crystallography reported herein indicate that multivalency effects underlie efficient dynein assembly and regulation. For a ternary complex of a 60-amino acid segment of dynein intermediate chain (IC) bound to two homodimeric dynein light chains Tctex1 and LC8, there is a 50-fold affinity enhancement for the second light chain binding. For a designed IC construct containing two LC8 sites, observed the 1000-fold enhancement reflects a remarkably pure entropic chelate effect of a magnitude commensurate with theoretical predictions. The lower enhancement in wild-type IC is attributed to unfavorable free energy changes associated with incremental interactions of IC with Tctex1. Our results show assembled dynein IC as an elongated, flexible polybivalent duplex, and suggest that polybivalency is an important general mechanism for constructing stable yet reversible and functionally versatile complexes.

FIGURE 1.

Schematic representation of cytoplasmic dynein and IC constructs used in this study. a, the cytoplasmic dynein core is shown as an assembly of six proteins: the N-terminal domain of IC (gray bars, predicted coiled-coils shown with red hashes) is natively disordered; the C-terminal domain of IC (gray spheres) is predicted to be ordered; and the three homodimeric light chains are Tctex1 (yellow), LC8 (green), and LC7 (blue). In mammals, the corresponding light chains are DYNLT, DYNLL, and DYNLRB, respectively (42). Also shown are the light intermediate chains (LIC, purple), the heavy chain (light blue), and a microtubule (orange). The motor region of dynein consists of the heavy chain subunits that form a ring of AAA+ domains (43) and a microtubule binding domain attached to the AAA+ ring by a flexible 15-nm coiled-coil stalk (44). An enlargement of the IC segment from residues 84–143 (dark blue brackets) has residues colored yellow and green, indicating the recognition sequences for Tctex1 and LC8, respectively. b, the three IC constructs IC_L, IC_TL, and IC_LL are IC residues 123–138, 84–143, and 84–143 with residues 111–120 replaced by a second copy of the LC8 binding sequence, respectively.

FIGURE 2.

Representative ITC data for IC constructs binding to Tctex1 and LC8. Thermograms (top panels) and binding isotherms (bottom panels) are shown for the titration of apo-IC_TL with (a) Tctex1 and (b) LC8, the titration of pre-bound IC_TL with (c) Tctex1 and (d) LC8, and (e) the titration of IC_LL with LC8. Solid lines correspond to the non-linear least squares fit for an A + B → AB binding model. Data were collected at 25 °C in 50 mm sodium phosphate, 50 mm sodium chloride, pH 7.5.

FIGURE 3.

Crystal structure of the ternary complexes of IC constructs with light chains. Semi-transparent surface and secondary structural elements are shown for (a) the WT IC·Tctex1·LC8 structure reported here (PDB entry 3FM7), (b) the IC·Tctex1·LC8 structure previously reported (PDB entry 2PG1) (15), and (c) the IC_LL·LC8·LC8 structure reported here (PDB entry 3GLW). In 2PG1, Tctex1 and LC8 form a small contact surface due to a bend in IC not observed in the structures reported here, implying that the IC linker remains flexible in the bound complex. The IC_LL·LC8·LC8 crystal had one LC8 chain and one-half of an IC_LL chain in the asymmetric unit, as discussed under ”Experimental Procedures“, the (d) center of the asymmetric unit and interpreted model of the IC_LL·LC8·LC8 structure are shown outlined in blue. For all figures Tctex1 is yellow, LC8 is green, and IC chains are black and white. Data collection and refinement statistics are given in supplemental Table S2. The figure was generated using PyMOL (45).

FIGURE 4.

Thermodynamic cycle for IC constructs binding to Tctex1 and LC8. Complete thermodynamic parameters on which this figure is based are given in Table 1. a, binding free energies ΔG⁰ (kcal/mol), and experimental ΔCp (kcal/mol/K) for Tctex1 and LC8 binding to IC_TL. The computed ΔCp_calc are given in parentheses following the experimental ΔCp. The difference in free energy change and heat capacity change between the second and first binding events are expressed as ΔΔG⁰ and ΔΔCp, respectively, and are shown in the center of the cycle. Similar binding parameters are shown for LC8 binding to IC_LL (b), which assumes the first IC_LL/LC8 binding event has thermodynamic binding parameters equal to the apo-IC_TL·LC8 binding event and LC8 binding to IC_L (c). All inferred data are labeled by asterisks.

FIGURE 5.

Changes in thermodynamic parameters between the first and second binding events (ΔΔG⁰, gray diamonds; ΔΔH⁰, black circles; and Δ(−TΔS⁰), white squares) for IC ternary complexes. Temperature dependence of the differences in association parameters between (a) LC8 with apo-IC_LL and LC8 with IC_LL/LC8, (b) Tctex1 with apo-IC_TL and Tctex1 with IC_TL/LC8, and (c) LC8 with apo-IC_TL and LC8 with IC_TL/Tctex1.