title: The intermediate chain of cytoplasmic dynein is partially disordered and gains structure upon binding to light-chain LC8.
abstract: The N-terminal domain of dynein intermediate chain, IC(1-289), is highly disordered, but upon binding to dynein light-chain LC8, it undergoes a significant conformational change to a more ordered structure. Using circular dichroism and fluorescence spectroscopy, we demonstrate that the change in conformation is due to an increase in the helical structure and to enhanced compactness in the environment of tryptophan 161. An increase in helical structure and compactness is also observed with trimethylamine-N-oxide (TMAO), a naturally occurring osmolyte used here as a probe to identify regions with a propensity for induced folding. Global protection of IC(1-289) from protease digestion upon LC8 binding was localized to a segment that includes residues downstream of the LC8-binding site. Several smaller constructs of IC(1-289) containing the LC8-binding site and one of the predicted helix or coiled-coil segments were made. IC(1-143) shows no increase in helical structure upon binding, while IC(114-260) shows an increase in helical structure similar to what is observed with IC(1-289). Binding of IC(114-260) to LC8 was monitored by fluorescence and native gel electrophoresis and shows saturation of binding, a stoichiometry of 1:1, and moderate binding affinity. The induced folding of IC(1-289) upon LC8 binding suggests that LC8 could act through the intermediate chain to facilitate dynein assembly or regulate cargo-binding interactions.
authors.affiliation: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
authors.forename: Afua
authors.initials: A
authors.lastname: Nyarko
authors.forename: Michael
authors.initials: M
authors.lastname: Hare
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Hays
authors.forename: Elisar
authors.initials: E
authors.lastname: Barbar
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D029721:Drosophila Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D004398:Dyneins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D006868:Hydrolysis; D008744:Methylamines / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D021122:Protein Subunits / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D014882:Water-Electrolyte Balance
chemical_list: D002352:Carrier Proteins; D029721:Drosophila Proteins; D008744:Methylamines; D010446:Peptide Fragments; D021122:Protein Subunits; D019286:Endopeptidase K; D004398:Dyneins; C005855:trimethyloxamine