Identification of key residues that regulate the interaction of kinesins with microtubule ends

Abstract

Kinesins are molecular motors that use energy derived from ATP turnover to walk along microtubules, or when at the microtubule end, regulate growth or shrinkage. All kinesins that regulate microtubule dynamics have long residence times at microtubule ends, whereas those that only walk have short end-residence times. Here, we identify key amino acids involved in end binding by showing that when critical residues from Kinesin-13, which depolymerises microtubules, are introduced into Kinesin-1, a walking kinesin with no effect on microtubule dynamics, the end-residence time is increased up to several-fold. This indicates that the interface between the kinesin motor domain and the microtubule is malleable and can be tuned to favour either lattice or end binding.

Keywords: Kinesin-1; Kinesin-13; MCAK; protein engineering; α4 helix.

Figure 1

Comparison of microtubule end residence for a purely translocating versus a regulating kinesin. (a) Distribution of microtubule end‐residence times observed for the Kinesin‐1, rkin430 (n = 273) and the Kinesin‐13, MCAK (n = 289). (b) Sequence alignment of the α4 helix of MCAK and rkin430. Asterisks indicate the positions at which Kinesin‐13 residues were substituted into Kinesin‐1 (red: positive, blue: negative, black: neutral). (c) Structure of MCAK (pdb: 5MIO) and the Kinesin 1, KIF5B (pdb: 4HNA) in complex with tubulin (green: kinesin motor domain, blue: α‐tubulin, orange: β‐tubulin). The substituted residues in the α4 helix are shown in magenta ball and stick format [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Substitution of Kinesin‐13 residues into the α4 helix of Kinesin‐1 increases its microtubule end residence. (a) Kymographs showing the interaction of the Kinesin‐1, rkin430 (green) and the Kinesin‐1 variant S266R (green) with a microtubule (magenta). Schematic highlighting kinesin interaction events is shown alongside each kymograph: events contained within the microtubule (blue) and events that reach the microtubule end (black). (b) Distribution of microtubule end‐residence times for wild‐type Kinesin‐1 and the variants S266R, G262K, N263E, a triple mutant (G262K/N263E/S266R) and S266A. (c) Relationship between translocation velocity and end residence for rkin430 and all variants. (d) End point of incubation of MCAK, rkin430 and variants of rkin430 with fluorescently labelled microtubules [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

C‐terminus of Kinesin‐13 α4‐helix is bulkier than Kinesin‐1. (a and b) Space‐fill models of the α4‐helix of (a) Kinesin‐1 (pdb: 4HNA) and (b) Kinesin‐13 (pdb: 5MIO). The Kinesin‐1 α4‐helix tapers towards the C‐terminus, whereas the Kinesin‐13 α4‐helix remains bulky. (c and d) End on view of the C‐terminus of the α4‐helix of (c) Kinesin‐1 and (d) Kinesin‐13 in complex with α/β‐tubulin. Kinesin (green), α‐tubulin (light blue), β‐tubulin (orange). The critical kinesin residues mutated in this study are coloured grey (neutral), red (positive charge) or dark blue (negative charge). (e) Alignment of the C‐terminal end of the α4‐helix for Kinesin‐1, MCAK (Kinesin‐13) and three other kinesins that regulate microtubule dynamics [Color figure can be viewed at http://wileyonlinelibrary.com]