The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

Abstract

Kinesin-5s are essential for forming the bipolar spindle during mitosis in most eukaryotes. The kinesin-5 motor domain contains conserved nucleotide and microtubule binding sites and mechanical elements to generate force. However, biochemical and biophysical studies have suggested that the mechano-chemistry of the kinesin-5 motor is very different from other kinesins. using cryo-electron microscopy and image reconstruction, we have calculated sub-nanometer resolution structures of microtubule-bound human kinesin-5 before and after nucleotide binding. Our structures reveal that, despite its mechanistic differences with conventional kinesin, kinesin-5 has the same coupled, nucleotide-dependent conformational changes as seen in conventional kinesins, including a ratchet-like docking of the neck linker, and simultaneous, parallel docking of the amino-terminal cover strand. These observations are supported by kinetic experiments that indicate a cooperative rearrangement of the kinesin-5-specific neck linker with the amino-terminal cover strand during the motor's ATPase cycle. In contrast to conventional kinesin however, our structures reveal a dramatic reorientation of Loop L5 - the binding site for allosteric inhibitors of kinesin-5-following ATP binding. This reorientation suggests that L5 is directly involved in controlling nucleotide binding by acting as an intra-molecular competitive inhibitor. Our structures indicate that allosteric inhibitors of human kinesin-5 bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Our data reveal the structural basis for kinesin-5 force generation that has evolved in the physiological context of the mitotic spindle.