Structure and interactions of subunits of cytoplasmic dynein.

摘要

Cytoplasmic dynein is a multimeric motor protein complex that is involved in retrograde transport along microtubules. It is composed of at least 12 subunits including a 530 kDa heavy chain subunit which binds microtubules, a 74 kDa subunit, IC 74 which plays a central role in the structure and functions of the complex and a 10 kDa subunit, LC8, which interacts with seemingly unrelated proteins. IC 74 is made up of two functionally independent domains; an N-terminal domain (IC 1-289) which associates with LC8 and a C-terminal domain (IC 290-642) which associates with the heavy chain subunit. The interaction with LC8 is of particular interest because of LC8's involvement with proteins of diverse biological functions. In this work the structure and interactions of IC 1-289 is characterized to show that this segment of the intermediate chain is partially unfolded, but undergoes a conformational change, adopting a helical conformation, upon binding LC8. The increase in helical structure was localized to a 118-residue segment downstream of the LC8 binding site, and which includes residues predicted to form a weak trimeric coiled-coil. Studies on the dynamics of residues in the predicted trimeric coiled-coil segment, monitored at 20°C and 5°C identified 6 residues, Glu 223, Glu 224, Gln 225, Lys 226, Gln 227 and Met 228 with a propensity to form helices. These residues possibly function as a "helix nucleation site" where helix formation is initiated and propagated to other segments of the intermediate chain to mediate folding upon LC8 binding. In a related study, the interaction between IC 1-289 and p150Glued , a subunit of a protein complex that is a regulator of cytoplasmic dynein, was probed with a number of spectroscopic tools. Unlike LC8, binding of p150Glued did not result in a change in the secondary structure of IC 1-289. Taken together these studies demonstrate a unique role for LC8 in stabilizing the intermediate chain subunit; a role which may also represent an important regulatory mechanism for the assembly of the dynein complex.