Abstract
The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLC(TPR)), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2(TPR) domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme.