Characterization of kinesin switch I mutations that cause hereditary spastic paraplegia

<div><p>Kif5A is a neuronally-enriched isoform of the Kinesin-1 family of cellular transport motors. 23 separate mutations in the motor domain of Kif5A have been identified in patients with the complicated form of hereditary spastic paraplegia (HSP). We performed in vitro assays on dimeric recombinant Kif5A with HSP-causing mutations in the Switch I domain, which participates in the coordination and hydrolysis of ATP by kinesin. We observed a variety of significantly reduced catalytic and mechanical activities as a result of each mutation, with the shared phenotype from each that motility was significantly reduced. Substitution of Mn²⁺ for Mg²⁺ in our reaction buffers provides a dose-dependent rescue in both the catalytic and ensemble mechanical properties of the S203C mutant. This work provides mechanistic insight into the cause of HSP in patients with these mutations and points to future experiments to further dissect the root cause of this disease.</p></div

Microtubule gliding velocity.

<p>Microtubule gliding velocity.</p

Effect of divalent cation on S203C microtubule gliding velocity.

<p>A) Microtubule gliding velocity of the S203C mutant in buffers with the divalent cations shown. The asterisk indicates that the velocity in Mn²⁺ is significantly greater than the velocity in Mg²⁺ (p<0.01), although still significantly lower than wild type in Mg²⁺ (p<0.01). Note the log scale of the Y-axis. B) Microtubule gliding velocity of the S203C mutant that was purified in the presence of Mg²⁺ (Mg-S203C) and Mn²⁺ (Mn-S203C). The single asterisk indicates that the velocity of Mg-S203C in Mn²⁺ is significantly greater than in Mg²⁺ (p<0.01), while the double asterisk indicates that the velocity of Mn-S203C in Mn²⁺ is significantly greater than in Mg²⁺ (p<0.01), and also significantly greater than the velocity of Mg-S203C in Mn²⁺ (p<0.05). C) Histograms of the data for S203C mutants in the conditions indicated. A similar number of microtubules (n = 90–110) were analyzed in each condition.</p

Molecular dynamics simulations of predicted altered kinesin structure.

<p>Images shown are the end point of a 10 ns simulation for wild type or mutant kinesin using the 4HNA structure as a starting point. Amino acids of interest are labeled in white. Intramolecular distances between potential bonding partners are shown in purple, with units of angstroms.</p

Effects of HSP mutations on the basal and MT-Stimulated ATPase rate.

<p>Effects of HSP mutations on the basal and MT-Stimulated ATPase rate.</p

MT affinity in the presence of AMPPNP.

<p>MT pelleting assays were performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180353#pone.0180353.g001" target="_blank">Fig 1</a>, except 5 mM AMPPNP was substituted in the reaction buffer. Experiments were repeated in a range of tubulin concentrations from 0 to 20 μM and the results of the densitometry analysis of the western blots produced are plotted for each Kif5A mutant. The curves shown are the best fit to the data using the exponential decay function in Origin 8 in a user free computational method. A) Curves of fraction Kif5A bound as a function of tubulin concentration for the wild type and mutant proteins. B-H) Individual Kif5A curves (WT or mutant as indicated) with mean +/- SD for each concentration of tubulin analyzed.</p