Abstract
In many eukaryotes, kinesin-5 motors are essential for mitosis, and small molecules that inhibit human kinesin-5 disrupt cell division. To investigate whether fungal kinesin-5s could be targets for novel fungicides, we studied kinesin-5 from the pathogenic fungus Ustilago maydis. We used cryo-electron microscopy to determine the microtubule-bound structure of its motor domain with and without the N-terminal extension. The ATP-like conformations of the motor in the presence or absence of this N-terminus are very similar, suggesting this region is structurally disordered and does not directly influence the motor ATPase. The Ustilago maydis kinesin-5 motor domain adopts a canonical ATP-like conformation, thereby allowing the neck linker to bind along the motor domain towards the microtubule plus end. However, several insertions within this motor domain are structurally distinct. Loop2 forms a non-canonical interaction with α-tubulin, while loop8 may bridge between two adjacent protofilaments. Furthermore, loop5 – which in human kinesin-5 is involved in binding allosteric inhibitors – protrudes above the nucleotide binding site, revealing a distinct binding pocket for potential inhibitors. This work highlights fungal-specific elaborations of the kinesin-5 motor domain and provides the structural basis for future investigations of kinesins as targets for novel fungicides.
Highlights
Fungi are significant and increasing mediators of pathogenesis, and cause challenges medically (e.g. Candida spp.; A. fumigatus), environmentally and economically (Bougnoux et al, 2018; Fisher et al, 2012)
To begin to understand whether Ustilago maydis kinesin-5 is a possible target for novel and specific fungicides, we determined the structures of two different motor domain constructs of this fungal kinesin – with and without its fungal-specific N-terminal extension bound to MTs using cryo-electron microscopy
Gero Steinberg (University of Exeter, UK), two Ustilago maydis kinesin-5 motor domain constructs – N+U. maydis kinesin-5 motor domain (UmKin5) and UmKin5 – with and without the N-terminal extension respectively, were PCR amplified and cloned into a pNIC28BsaI vector (Structural Genomics Consortium, Oxford), and the recombinant His6-tagged monomeric constructs were expressed in BL21*(DE3) Escherichia coli cells
Summary
Fungi are significant and increasing mediators of pathogenesis, and cause challenges medically (e.g. Candida spp.; A. fumigatus), environmentally (e.g. ash dieback-causing H. fraxinea) and economically (e.g. rice blast, M. oryzae; honeybee colony collapse, Nosema spp.) (Bougnoux et al, 2018; Fisher et al, 2012). Kinesins are microtubule (MT)-based ATP-driven motors that have many important roles in eukaryotes. This includes the essential activities of several members of the superfamily in cell division (Cross and McAinsh, 2014). Kinesin-5 motors are important for mitosis in many organisms and, for example, functional disruption of kinesin-5s in fungi and vertebrates prevents formation of the bipolar spindle (Goulet and Moores, 2013). Mechanistic information is lacking about the extent of conservation of kinesin-5 molecular mechanism across eukaryotes, and whether any structural or mechanistic differences could be exploited to selectively inhibit mitosis in pathogenic organisms.
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