Fig. 5: Plasmodium kinesin-8B neck linker is required for both motility and depolymerase activities.
From: Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission
a Schematic of MD and MDΔNL constructs of Pbkinesin-8B and Pfkinesin-8B. Motor domains are coloured in green and blue, respectively; neck linker sequences are coloured in red. b Kinesin-8B-MDΔNL constructs exhibit reduced MT-stimulated ATPase activity compared to kinesin-8B-MD (Pb:GMPCPP-MT, Pf:paclitaxel-stabilised MT). Data (n = 3 for each point) was fitted using Michaelis-Menten equation (mean ± SD), from which the Kcat and KM were calculated in Prism9. c Kinesin-8B-MDΔNL construct exhibit no significant gliding activity. Paclitaxel-stabilised MTs were used. Error bars represent the mean ± SD and individual measurements are also plotted with coloured points. Ordinary one-way ANOVA was performed in Prism. Significance values are displayed as asterisks, ****p-values < 0.0001; ns not significant, p = 0.5803 (no-kinesin-8B vs. Pbkinesin-8B-MDΔNL) and 0.0831 (no-kinesin-8B vs. Pfkinesin-8B-MDΔNL). NPbkinesin-8B-MD = 36 MTs. NPbkinesin-8B-MDΔNL = 67 MTs. NPfkinesin-8B-MD = 104 MTs. NPfkinesin-8B-MDΔNL = 77 MTs. Nno kinesin-8B = 24 MTs. d Paclitaxel-stabilised-MT depolymerisation rate (nm/s) for Pbkinesin-8B-MDΔNL and Pfkinesin-8B-MDΔNL in the presence of ATP compared to Pbkinesin-8B-MD and Pfkinesin-8B-MD and a no-kinesin control. Error bars represent the mean ± SD and individual measurements are also plotted with coloured points. Ordinary one-way ANOVA was performed in Prism. Significance values are displayed as asterisks, ****p-values < 0.0001; ns not significant, p = 0.0741 (no-kinesin-8B vs. Pbkinesin-8B-MDΔNL) and 0.3518 (no-kinesin-8B vs. Pfkinesin-8B-MDΔNL). NPbkinesin-8B-MD = 97 ends. NPbkinesin-8B-MDΔNL = 91 ends, NPfkinesin-8B-MD = 103 ends. NPfkinesin-8B-MDΔNL = 75 ends, Nno kinesin-8B = 85 ends. Data for Pfkinesin-8B-MD are replotted from Fig. 2B, while data for Pbkinesin-8B-MD were collected in parallel with mutant activity measurement using the same MT prep on the same day; differences in depolymerisation rates between different experiments most likely relate to different MT stability between different preps.
