Cytoplasmic dynein is an ATP-dependent, microtubule-based molecular motor involved in the positioning and trafficking of cellular cargo. The cargo binding sub-domain of dynein contains the natively disordered intermediate chain (IC) and the homodimeric light chains (Tctex1, LC8 and LC7). The structure and stoichiometry of this complex, how the light chains interact with natively disordered IC and the role of the light chains in dynein assembly is not clear. The focus of these studies is to elucidate the interactions between IC and the light chains using dynamical, thermodynamical and structural biophysics techniques.
In chapters 2 and 3 we show changes in LC8 internal dynamics are important for partner binding. Furthermore we show the two binding sites on LC8 are allosteric, with both structural and dynamical changes apparent between binding events. These results demonstrate the importance of protein dynamics in LC8 binding.
In chapter 4 we determined the structure of the IC•Tctex1•LC8 complex and show multivalency between Tctex1 and LC8 results in a 50-fold affinity enhancement for IC binding despite no direct Tctex1•LC8 interaction. Interestingly, a purely entropic 1000-fold binding affinity enhancement exists for a designed IC construct containing two LC8 sites instead of a Tctex1 and LC8 site. These results demonstrate Tctex1 and LC8 multivalency is tailored to fit the association needs of the system instead of maximizing potential binding energy.
In chapter 5 we determine the structure of the IC•LC7 complex and show the LC7 recognition sequence on IC proposed in the literature is wrong, instead the LC7 recognition sequence overlaps residues previously thought to be the IC•IC self-association domain. Furthermore, we show Tctex1 and LC8 have a negligible effect on net IC•LC7 binding affinity, despite evidence that Tctex1 and LC8 affect the nature of the IC•LC7 interaction. Thus this work provides the first atomic-level structure of the IC•LC7 complex, corrects decade long misconceptions about IC•LC7 binding and demonstrates an interconnection between the light chains for IC association.
Individually these results provide detailed descriptions of IC and the light chains; together they strongly suggest the light chains structurally regulate IC assembly into the mature cargo binding sub-domain of dynein.
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