This proposal focuses on interactions of two subunits of cytoplasmic dynein: LC8, a 10 kDa light chain, and IC74, the 74 kDa intermediate chain. We have overexpressed these subunits from Drosophila melanogaster and characterized their interactions in vitro by affinity methods, limited proteolysis, and circular dichroism.
Our first aim i s to identify the residues on each subunit which are responsible for the interaction. We have localized the binding site to the segment in the vicinity of K130 on IC74. We will use NMR to identify interactions at the atomic scale, with constructs of IC74 designed and prepared for this purpose. Identification of the binding residues by NMR involves the determination of local changes in chemical shifts, relaxation rates and line widths in heteronuclear NMR experiments.
Our second aim i s to characterize the conformational changes in IC74 upon binding to LC8. We have observed that binding to LC8 causes a dramatic change in circular dichroism spectra of the N-terminal domain of IC74. These changes are consistent with greater ordering in one or both of the coiled-coil regions predicted to be in IC74. Since the typical function of the coiled-coil motif is to nucleate the formation of multi-subunit protein complexes, greater ordering in these regions would increase the propensity of IC74 to bind to other subunits of dynein, and LC8 binding to IC74 would be clearly implicated as an initiating factor in the assembly of the complex. We will use NMR techniques to study the dynamics of bound and free IC74 constructs, and locate the segments involved in the increased order. We will also use hydrogen exchange mass spectrometry to examine the effect of LC8 binding on increasing structure and stability of IC74 by measurements of changes in solvent accessibility between bound and free. We have previously demonstrated the efficacy of this versatile technique in a study of the dynamics of monomeric and dimeric LC8.The methods that will be developed in this work will be increasingly important in our ongoing investigation of the structural and dynamic basis for the assembly and function of cytoplasmic dynein. These investigations will shortly encompass LC14 and other subunits of dynein, the accessory complex dynactin, and dynein cargo. With a collaborator who is an expert in dynein biology, we plan to examine the functional relevance of our findings in Drosophila.
