A Mechanism for Microtubule Depolymerization by KinI Kinesins Carolyn A. Moores, Ming Yu, Jun Guo, Christophe Beraud, Roman Sakowicz, Ronald A. Milligan  Molecular Cell  Volume 9, Issue 4, Pages 903-909 (April 2002) DOI: 10.1016/S1097-2765(02)00503-8

Figure 1 Interaction of pKinI with Microtubules through Its ATPase Cycle (A) 5 μM taxol-stabilized microtubules were incubated for 30 min at room temperature with 10-fold less pKinI motor and ATP, and the products were analyzed by cosedimentation. Tubulin was released from microtubules to the supernatant in the presence of substoichiometric quantities of pKinI motor. (B) Increasing concentrations of pKinI motor were incubated with microtubules in the presence of (i) no nucleotide, (ii) AMPPNP, (iii) ADP.AlF4, and (iv) ADP. The mixture was separated into supernatant and pellet fractions by ultracentrifugation. In each case, the concentrations of polymerized tubulin were 5 μM and of pKinI were (i) 0.93, 2.8, and 6.53 μM; (ii) 0.7, 3.2, and 6.4 μM; (iii) 0.83, 2.5, and 6.25 μM; and (iv) 1.01, 3.02, and 6.04 μM. (C) The pellet fraction from a cosedimentation experiment of pKinI incubated with AMPPNP and taxol-stabilized microtubules was resuspended in buffer and visualized by negative stain electron microscopy. Binding of the pKinI motors was observed along the wall of the microtubule every 80 Å. Scale bar = 200 Å. (D) Subtilisin-treated taxol-stabilized microtubules were analyzed by SDS-PAGE and by Western blotting, probed with antibodies against anti-β-tubulin and anti-α-tubulin. Full-length tubulins run together, but lower molecular weight bands result from removal of the acidic C termini from α- and β-tubulin. (E) The microtubule products of subtilisin cleavage (2 μM) were incubated with pKinI motor (5 μM) (i) in the absence of nucleotide and (ii) with AMPPNP. Microtubule-bound and free protein were separated as done previously and analyzed by SDS-PAGE. In the absence of nucleotide, binding to the microtubules by the pKinI motor is the same +/− subtilisin treatment. In the presence of AMPPNP, tubulin is released to the supernatant from untreated microtubules, while no depolymerization was seen from the subtilisin-treated microtubules. Molecular Cell 2002 9, 903-909DOI: (10.1016/S1097-2765(02)00503-8)

Figure 2 pKinI Captured in the Act of Depolymerization (A and B) Images of tubulin rings induced by pKinI-AMPPNP binding. Arrowhead in (B) indicates a distorted ring, demonstrating that the rings are composed of a single protofilament. Scale bar = 400 Å. (C) Grayscale image of 13-fold averaged 2-dimensional map of pKinI-AMPPNP-induced tubulin rings. Scale bar = 45 Å. (D) Contour map of pKinI-induced tubulin ring and (E) a contoured section through the wall of a 3-dimensional map of a microtubule decorated with monomeric conventional kinesin. A motor domain is labeled * in each. Molecular Cell 2002 9, 903-909DOI: (10.1016/S1097-2765(02)00503-8)

Figure 3 Model for Microtubule Depolymerization by the KinI Motor (A) Atomic model of the interaction between a kinesin motor and GDP-tubulin. The insertion point to the N terminus of the kinesin motor core is marked with a green *, and the approximate position for the start of the β-tubulin C terminus is marked with a pink *. The region in red shows sequence divergence among kinesins and could be important in contributing to KinI motor depolymerizing activity. α4, the motor relay helix, is shown in orange. The figures were prepared using MOLSCRIPT (Kraulis, 1991) and Raster3D (Merritt and Bacon, 1997). PDB coordinates of conventional kinesin are 3KIN and GDP-tubulin 1FFX. (B) Model for the stages of KinI motor ATPase-coupled microtubule depolymerization. The KinI motor binds to microtubule ends in a similar way to other kinesin motors, releasing bound ADP (1), but when ATP binds (2), a conformational change in the tubulin dimer is induced. This conformational change requires the presence of the acidic C terminus of β-tubulin, which is shown in black. This could involve KinI interaction with the β-tubulin peptide on the dimer to which the motor is bound (i) or could involve tugging on the β-tubulin C terminus of an adjacent dimer (ii). Subsequent steps are required to recycle the KinI motor, a process presumably involving tubulin dimer release from the protofilament (3) and motor-tubulin dissociation (4), completing the ATP hydrolysis cycle (5). Molecular Cell 2002 9, 903-909DOI: (10.1016/S1097-2765(02)00503-8)