Altered Mechanical Properties of Kinesins with Mutations that Cause Hereditary Spastic Paraplegia

Abstract

Hereditary Spastic Paraplegias (HSPs) are a genetically and clinically heterogeneous set of neurodegenerative diseases that all share an axonopathy of the corticospinal tract. To date, there are at least 70 distinct genetic loci that, when mutated, cause HSP in humans. One of those loci is the neuronally enriched kinesin transport motor, Kif5A. Of particular interest to this study is that 22 of the 25 mutations in Kif5A that have been shown to cause HSP are in the motor domain - the portion of the kinesin protein that has the dual responsibility of interacting with the microtubule and carrying out the ATPase cycle. Perhaps unsurprisingly, most of the mutations cluster to either the microtubule binding site or the nucleotide binding pocket. We have recombinantly-expressed Kif5A with each of the separate HSP-causing mutations and have performed a series of well-characterized in vitro assays (ATPase, stopped flow, microtubule affinity, microtubule gliding, single molecule motility) to better understand the altered catalytic properties that result from each mutation. In addition, we have performed molecular dynamics simulations to model the altered structural properties of each of the mutant proteins. We find that all mutations are loss-of-function mutations, and that there are separable functional deficits for each mutation that limit kinesin's ability to functionally transport cellular cargoes.