Transducer Residues are Thermodynamically Coupled in the Kinesin-5 Motor Domain

Abstract

Motor proteins coordinate the activities of nucleotide hydrolysis and polymer binding to move along cellular tracks. In kinesin and myosin motor domains, the sites responsible for these two activities are located on opposite faces of a core beta-sheet. Three beta-strands and a mobile loop transduce information between the active and polymer-binding sites and, thus, are collectively termed the transducer. Herein we demonstrate residues in the core beta-sheet and the L5 loop are thermodynamically coupled. Two methods were employed in this study. Probabilistic methods to analyze evolutionarily correlations between residues in protein families identified coupling between transducer residues. Second, thermodynamic linkage between M115 in loop-5 and L263 in beta-7 in the human kinesin-5 motor domain was established using double mutant cycle analysis for wildtype, two single mutants, and the corresponding double mutant protein. The resulting changes in free energy were calculated from experimentally measured kinetic parameters and determined to be non-additive. While conformational changes of the beta strands and mobile loop have been observed in structural investigations of kinesins and myosins, these experiments establish energetic and evolutionary coupling of these distinct motifs. We conclude that loop-5 and the beta-strands are important kinesin building blocks that act in concert during mechanotransduction. This work was funded by NIH (GM097350 to SK).