Abstract
KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7–3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.
Highlights
KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers
To elucidate conformational changes related to the microtubule-bound KIF14 motor domain ATPase cycle, and their possible modulation by the neck-linker or a partner motor domain, we determined the Cryo-electron microscopy (cryo-EM) structures of microtubule (MT) complexes of five distinct KIF14 constructs in four nucleotide conditions
We have solved the near-atomic resolution cryo-EM structures of five distinct KIF14 protein constructs bound to microtubules, in four nucleotide conditions mimicking key steps of the KIF14 ATPase cycle
Summary
KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. KIF14 possesses a highly conserved catalytic motor or head domain that contains nucleotide and microtubule binding sites, and where ATP hydrolysis is coupled to the generation of mechanical work. Much is known regarding the mechanism of action of motile kinesins, and in particular of its founding member kinesin-1, most structural information of kinesin-microtubule complexes is still of limited resolution As a consequence, it is still not fully clear how conformational changes are coupled to distinct steps of the ATP hydrolysis cycle or how the two heads of a kinesin dimer may coordinate their activities. We implemented an analysis procedure to separate coexisting conformations of the kinesin motor bound to the microtubule helical assembly With this procedure, we determined the structure of the two motor domains of KIF14 dimers bound to the microtubule (two-headsbound state) at high resolution. Our structural data provide a comprehensive molecular understanding of how kinesin ATPase cycle is coordinated with plusend-directed movement along microtubules
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