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Title: 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.

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930672
Report Number(s):
BNL-81175-2008-JA
Journal ID: ISSN 0027-8424; PNASA6; TRN: US200901%%174
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proceedings of the National Academy of Sciences of the USA; Journal Volume: 104
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CELL CONSTITUENTS; CHAINS; HYPOTHESIS; MITOSIS; MOTORS; PROTEINS; THERMODYNAMICS; TRANSPORT; national synchrotron light source

Citation Formats

Williams,J., Roulhac, P., Roy, A., Vallee, R., Fitzgerald, M., and Hendrickson, W. Structural and Thermodynamic Characterization of a Cytoplasmic Dynein Light Chain-Intermediate Chain Complex. United States: N. p., 2007. Web. doi:10.1073/pnas.0703614104.
Williams,J., Roulhac, P., Roy, A., Vallee, R., Fitzgerald, M., & Hendrickson, W. Structural and Thermodynamic Characterization of a Cytoplasmic Dynein Light Chain-Intermediate Chain Complex. United States. doi:10.1073/pnas.0703614104.
Williams,J., Roulhac, P., Roy, A., Vallee, R., Fitzgerald, M., and Hendrickson, W. Mon . "Structural and Thermodynamic Characterization of a Cytoplasmic Dynein Light Chain-Intermediate Chain Complex". United States. doi:10.1073/pnas.0703614104.
@article{osti_930672,
title = {Structural and Thermodynamic Characterization of a Cytoplasmic Dynein Light Chain-Intermediate Chain Complex},
author = {Williams,J. and Roulhac, P. and Roy, A. and Vallee, R. and Fitzgerald, M. and Hendrickson, W.},
abstractNote = {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.},
doi = {10.1073/pnas.0703614104},
journal = {Proceedings of the National Academy of Sciences of the USA},
number = ,
volume = 104,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • The human light chain of the motor protein dynein, Dnlc2A, is also a novel TGF-{beta}-signaling component, which is altered with high frequency in epithelial ovarian cancer. It is an important mediator of dynein and the development of cancer, owing to its ability to bind to the dynein intermediate light chain (DIC) IC74 and to regulate TGF-{beta}-dependent transcriptional events. Here we report the 2.1-A crystal structure of Dnlc2A using single anomalous diffraction. The proteins form a homodimer in solution and interact mainly through the helix {alpha}{sub 2}, strand {beta}{sub 3}, and the loop following this strand in each protein to generatemore » a 10-stranded {beta}-sheet core. The surface of the {beta}-sheet core is mainly positively charged and predicted (by software PPI-Pred) to be the site that interacts with other partners. At the same time, the residues 79-82, 88, and 90 of each molecule formed two holes in the core. Residue 89 of each molecule, which is crucial for the DIC binding function of Dnlc2A, is within the holes. On the basis of these observations, we propose that the homodimer is the structural and functional unit maintained by hydrogen bonding interactions and hydrophobic packing, and that the patch of the surface of the {beta}-sheet core is the main area of interaction with other partners. Furthermore, the two holes would be the key sites to interact with IC74.« less
  • Dyneins are a group of microtubule-activated ATPases that serve to convert chemical energy into mechanical energy. They have been divided into two large subgroups, namely the axonemal and cytoplasmic dyneins. Cytoplasmic dynein has been implicated in a variety of other forms of intracellular motility, including retrograde axonal transport, protein sorting between apical and basolateral surfaces, and redistribution of organelles like endosomes and lysosomes. Our report is the first chromosomal localization of the human ctyoplasmic dynein heavy chain (DNECL). 7 refs., 1 fig.
  • No abstract prepared.
  • Dynein light chain LC8 is the smallest subunit of the dynein motor complex and has been shown to play important roles in both dynein-dependent and dynein-independent physiological functions via its interaction with a number of its binding partners. It has also been linked to pathogenesis including roles in viral infections and tumorigenesis. Structural information for LC8-target proteins is critical to understanding the underlying function of LC8 in these complexes. However, some LC8-target interactions are not amenable to structural characterization by conventional structural biology techniques owing to their large size, low solubility, and crystallization difficulties. Here, we report magic-angle spinning (MAS)more » NMR studies of the homodimeric apo-LC8 protein as a first effort in addressing more complex, multi-partner, LC8-based protein assemblies. We have established site-specific backbone and side-chain resonance assignments for the majority of the residues of LC8, and show TALOS+-predicted torsion angles ø and ψ in close agreement with most residues in the published LC8 crystal structure. Data obtained through these studies will provide the first step toward using MAS NMR to examine the LC8 structure, which will eventually be used to investigate protein–protein interactions in larger systems that cannot be determined by conventional structural studies.« less