Structural and thermodynamic characterization of a cytoplasmic dynein light chain-intermediate chain complex

Abstract

Cytoplasmic dynein is a microtubule-based motor protein complex that plays important roles in a wide range of fundamental cellular processes, including vesicular transport, mitosis, and cell migration. A single major form of cytoplasmic dynein associates with membranous organelles, mitotic kinetochores, the mitotic and migratory cell cortex, centrosomes, and mRNA complexes. The ability of cytoplasmic dynein to recognize such diverse forms of cargo is thought to be associated with its several accessory subunits, which reside at the base of the molecule. The dynein light chains (LCs) LC8 and TcTex1 form a subcomplex with dynein intermediate chains, and they also interact with numerous protein and ribonucleoprotein partners. This observation has led to the hypothesis that these subunits serve to tether cargo to the dynein motor. Here, we present the structure and a thermodynamic analysis of a complex of LC8 and TcTex1 associated with their intermediate chain scaffold. The intermediate chains effectively block the major putative cargo binding sites within the light chains. These data suggest that, in the dynein complex, the LCs do not bind cargo, in apparent disagreement with a role for LCs in dynein cargo binding interactions.

Fig. 1.

Ribbon diagram of the dynein complex. (A) TcTex1 (cyan) and LC8 (wheat) homodimers are associated with two IC peptides (residues 104–138; magenta). Each IC peptide binds to one side of each LC dimer producing a 2:2:2 complex. Residues 112–124 of the IC contact TcTex1, and residues 129–137 contact LC8. Two views are shown, rotated by 90° about the vertical axis. (B) Experimental electron density of the IC peptide in green on the electrostatic surface of LC8 and TcTex1 (54) in stereo.

Fig. 2.

Common features of the LC-target interactions. (A) Stereoview of TxTex1 (cyan ribbon) and the IC peptide (stick representation). The IC residue, D121, highlight by the red oval is equivalent to the invariant glutamine found in all LC8 target peptides. The green area indicates the hydrophobic contacts between the IC and TcTex1. This region also maps to an equivalent site in LC8. The pink highlighted regions indicate additional hydrophobic sites that may contribute to target specificity. Mutation of L112 to alanine abrogated the TcTex1–IC interaction (24) (A. Dawn and J.C.W., unpublished data). (B) Superposition of β-strands of TcTex1 (cyan) and LC8 (wheat). Although TcTex1 and LC8 are structurally similar, TcTex1 is generally extended compared with LC8. This extension results in additional contacts for the IC peptide. Also note that the α2-helix bends away from the IC peptide. (C) Stereoview of LC8 (wheat ribbon) and the IC peptide (stick representation). The invariant glutamine is highlighted in red. The hydrophobic binding pockets are highlighted in green.

Fig. 3.

Thermodynamic analysis. (A) SUPREX curves for 85 μM LC8 (filled circles), 77 μM LC8 in the presence of 442 μM IC_MONO (open circles), 77 μM LC8 in the presence of 442 μM nNOS peptide (filled squares), and 130 μM LC8 in the presence of 374 μM IC_MONO and 130 μM TcTex1 (open triangles). All curves shown were subjected to an H/D exchange time of 45 min. (B) The −RT ln[((<k_int>t/0.693) − 1)/((nⁿ/2ⁿ⁻¹)[P]ⁿ⁻¹)] (i.e., −ΔG_app) versus C_SUPREX^1/2 plots obtained for LC8 (filled circles), LC8 in the presence of IC_MONO (open circles), LC8 in the presence of nNOS peptide (filled squares), and LC8 in the presence of IC_MONO and TcTex11 (open triangles). Protein and peptide concentrations are the same as in A. (C) SUPREX curves for 10 μM LC8 (open circles), 9 μM LC8 in the presence of 454 μM IC_MONO (open diamonds), 8 μM LC8 in the presence of 417 μM IC_MONO and 8 μM TcTex1 (open squares), and 13 μM LC8 in the presence of 35 μM IC_DIMER (open triangles). All curves shown were subjected to an H/D exchange time of 45 min.

Fig. 4.

Potential role of the LCs in cytoplasmic dynein. (A) Model based on the structure and thermodynamic characterization. A cartoon of the dynein motor complex (HC, IC, and LCs) bound to a microtubule (green/yellow) is shown in the upper right corner. The boxed region is the N-terminal half of the IC, part of which includes the IC–LC complex presented herein. The structure represents a probable state of the dynein complex “life cycle.” The prevalent model for the LCs in terms of cargo transport is presented in box 1. Nearly all LC8 targets encode a canonical sequence containing an invariant glutamine (see text) and bind to the same site as the IC. Because the LCs are homodimeric, one binding site can bind to the IC, and the other can bind to a putative LC cargo. However, the ICs and many of the LC targets are dimeric. Thus, they act as bivalent ligands for the bivalent LCs and thus gain affinity through energy additivity as demonstrated by our SUPREX measurements. Because many of the targets identified to bind to the LCs are dimeric, they can compete with the IC for LC binding as shown in box 2. In this model, the dimeric LC targets compete with the IC for LC binding. This would decouple the LCs from the dynein motor and negate their role in cargo transport, in disagreement with the current model. Alternatively, a posttranslation modification of the LCs or IC could lead to their dissociation. If the local concentration is less than the LC dimerization constant, the LCs could become monomeric (box 3). Monomeric LC8 cannot bind the dimeric IC (C. M. Lightcap and J.C.W., unpublished data). (B) Schematic of the dynein IC. The C-terminal region of the IC contains a WD domain that binds to the dynein HCs. The N-terminal region contains a strongly predicted coiled coil region, a serine rich region and variable splice sites. This N-terminal region of the IC, which is strongly predicted to be intrinsically unstructured, binds to p150^Glued and the LCs. Phosphorylation of the IC at Ser-84 blocks the dynein–dynactin interaction.