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.
Highlights
Molecular genetic analyses in D. melanogaster indicate that intermediate chain (IC) serves an essential function [3], consistent with its multiple roles in dynein assembly, regulation, and binding to cargo
In vitro studies of various segments of N-IC indicate that it is natively disordered and monomeric [11, 12]; two N-IC chains bind to both Tctex1 and LC8, and within each chain the 10 –12-amino acid recognition sequence undergoes a disorder-to-order transition [13] to form a strand incorporated into a -sheet at the light chain dimer interface [14, 15]
In vivo studies [18] of various truncations of N-IC show that an IC lacking the light chains recognition sequence binds less efficiently to p150Glued and is less effective at inhibiting dyneinbased transport than a longer IC containing the light chains recognition sequence and the second predicted coiled-coil. In such experiments, overexpressed IC is presumed to bind free dynactin and competes with endogenous dynein IC resulting in perturbed microtubule organization and centrosome integrity
Summary
Molecular genetic analyses in D. melanogaster indicate that IC serves an essential function [3], consistent with its multiple roles in dynein assembly, regulation, and binding to cargo. Apo-ICTL binds Tctex1 or LC8 with a similar affinity (Kd of 8 M), and a ⌬Cpexp of Ϫ0.42 and Ϫ0.26 kcal/mol/K, respectively (see Table 1 and supplemental Fig. S2, b and c).
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