Vik1 modulates microtubule-Kar3 interactions through a motor domain that lacks an active site

Abstract

Conventional kinesin and class V and VI myosins coordinate the mechanochemical cycles of their motor domains for processive movement of cargo along microtubules or actin filaments. It is widely accepted that this coordination is achieved by allosteric communication or mechanical strain between the motor domains, which controls the nucleotide state and interaction with microtubules or actin. However, questions remain about the interplay between the strain and the nucleotide state. We present an analysis of Saccharomyces cerevisiae Kar3/Vik1, a heterodimeric C-terminal Kinesin-14 containing catalytic Kar3 and the nonmotor protein Vik1. The X-ray crystal structure of Vik1 exhibits a similar fold to the kinesin and myosin catalytic head, but lacks an ATP binding site. Vik1 binds more tightly to microtubules than Kar3 and facilitates cooperative microtubule decoration by Kar3/Vik1 heterodimers, and yet allows motility. These results demand communication between Vik1 and Kar3 via a mechanism that coordinates their interactions with microtubules.

Figure 1. Bar Diagrams of the Predicted Structural Domains of Full-length Kar3, Cik1, and Vik1

The coiled-coil regions (CC) were predicted by PAIRCOIL (Berger et al., 1995). The regions of each protein that were used to make the truncated versions of Kar3, Cik1, and Vik1, as well as the Kar3MD and Vik1MHD constructs are indicated next to each bar diagram.

Figure 2. The Structure of Vik1 and Its Comparison to Kar3

(A) A ribbon representation of the structure of the C-terminal globular domain of Vik1 (left), including a short stretch of the coiled-coil forming region (neck), is shown beside the motor domain of Kar3 (right) (PDB accession: 3KAR) (Gulick et al., 1998). The construct used for crystallization consisted of residues Thr³⁵³ to Thr⁶⁴⁷ from Vik1, however the model has been truncated at Cys⁶⁴⁰ due to disorder of the C-terminus. Thr³⁵³ is preceded by three residues, Gly³⁵⁰, Ala³⁵¹, and Ser³⁵², respectively, at the N-terminus of the molecule, which are a result of the affinity tag used for protein purification. Poor electron density existed for several loops, and were not built into the final model. These lie between residues Tyr⁴⁹⁷ and Asp⁵⁰³, Asp⁵³⁷ and Ser⁵⁴⁵, Ser⁵⁵⁹ and Pro⁵⁶⁷, and Lys⁵⁹³ and Ser⁵⁹⁵. Residues between Ser⁴⁸⁵ and Ser⁴⁹⁰ are also located on a loop that is not well-ordered, however, the electron density was of sufficient quality to build this region into the model when an occupancy of 0.5 was applied. (B) Topology diagrams for the Vik1 (left) and Kar3 (right) motor domains were generated with TopDraw based on topology analyses by the TOPS server (Westhead et al., 1999; Bond, 2003). The diagrams are labeled and colored to match the structures in (A). (C) Electrostatic surface representation of the nucleotide-binding pocket of Kar3MD (upper right) and the analogous region of Vik1MHD (upper left) with MgADP superimposed after alignment of the α-carbons of the Vik1MHD (Gly³⁷³ to Cys⁶⁴⁰) onto those of the Kar3MD structure. ADP is shown as yellow sticks and Mg²⁺ as a green sphere. Mg²⁺ is obscured by the protein surface in the Vik1MHD figure. The electrostatic surface potential was generated in Pymol using APBS (Baker et al., 2001; DeLano, 2002). A ribbon representation of the P-loop (including relevant side chains) of Kar3 and its interaction with MgADP is shown (lower right). The analogous loop of Vik1 is shown with MgADP superimposed based on the overall alignment of Vik1MHD onto the Kar3MD structure (lower left). All structure figures were generated with Pymol (DeLano, 2002).

Figure 3. Microtubule Binding by Vik1MHD, SeMetVik1MHD, Kar3MD, and Kar3/Vik1

(A and C) Microtubule•motor cosedimentation was performed to compare the binding of 50 nM Vik1MHD, SeMetVik1MHD, Kar3MD, and (B and C) Kar3/Vik1 at three different nucleotide conditions. The data were plotted as the fraction of motor partitioning to the pellet as a function of microtubule concentration and fit to quadratic equation 2, providing the constants in C. Data are reported as mean +/− SEM. Final concentrations: 0–3 μM tubulin polymer, 40 μM Taxol, and ± 0.1U/ml Apyrase, or 2 mM MgAMPPNP, or 2 mM MgADP. (D and E) Comparison of microtubule-binding surfaces of Vik1MHD and Kar3MD. The shown view is rotated 180° from Figure 2A. Putative microtubule-binding elements of Kar3MD (E) and the analogous regions of Vik1 (D) whose structural properties are significantly different are shown in orange.

Figure 4. Kar3/Vik1 Is a Kinesin-14 Heterodimeric Motor

(A) Kar3/Vik1 minus-end-directed motility in the presence of MgATP. Arrowheads denote the bright microtubule minus end, and the asterisks (*), the dim, leading microtubule plus end. Scale bar = 5μm. The table compares the microtubule gliding promoted by Kar3/Vik1, Kar3/Cik1, and squid Kinesin-1. (B) The steady-state ATPase kinetics of Kar3/Vik1 and Kar3/Cik1 as a function of microtubule and MgATP concentrations. Upper panel final concentrations: 0.82 μM Kar3/Vik1 or 1.1 μM Kar3/Cik1, 0–42 μM tubulin polymer, 40 μM Taxol, and 1 mM [α³²P] MgATP. Lower panel final concentrations: 0.85 μM Kar3/Vik1 or 1 μM Kar3/Cik1, 20 μM tubulin polymer, 40 μM Taxol, and 0–1 mM [α³²P] MgATP. The table shows the steady-state parameters of Kar3/Vik1 and Kar3/Cik1 in comparison to the Kar3MD (Mackey and Gilbert, 2003). (C) ATP-dependent Kar3/Vik1 and Kar3/Cik1 promoted microtubule shortening. Microtubule•motor complexes were preformed in the presence of 1 mM MgAMPPNP and imaged at t = 0, column 1. MgATP at 1.5 mM plus an ATP regeneration system initiated microtubule shortening (Sproul et al., 2005). Column 3 is the merge of t = 0 and the elapsed time (middle column) to show microtubule shortening (red) in comparison to the original length. Polarity-marked microtubules were identified from microtubule•motor populations at both 25 and 50 nM motor incubated with 500 nM microtubules in the presence of MgATP. (D) Increased motor binding to microtubules stabilizes the microtubule lattice against shortening. Upper panel: Kar3/Cik1 and Kar3/Vik1 rates of microtubule shortening plotted as a function of increasing motor concentration. Lower panel: The percentage of microtubules that showed Kar3/Cik1 or Kar3/Vik1-promoted ATP-dependent shortening plotted as a function of motor concentration. Data are reported as mean +/− SEM.

Figure 5. Immunolocalization of Kar3/Vik1, Kar3/Cik1, Kar3MD, and Vik1MHD

Microtubule•motor complexes were preformed in the presence of MgAMPPNP. Final concentrations: 500 nM tubulin polymer, 40 μM Taxol, and 1 mM MgAMPPNP. Rows 1 and 2 represent magnification of a section of the field, whereas the remaining rows show individual microtubules at a higher magnification (scale bars = 5 μm). The microtubule seed (arrowhead) marks the microtubule minus end and (*) denotes the dim microtubule plus end. The first column of each row shows the rhodamine-labeled microtubules, and the second column, the immunofluorescence of affinity-purified Vik1MHD antibodies. The third column is the merge of the two channels to show the co-localization. The table presents the summary of microtubule localization events scored for the three motors using affinity-purified antibodies to the Kar3MD or the Vik1MHD (Figure S1).

Figure 6. Comparison of the Neck Orientations of Vik1 and Ncd

The view shown has been rotated 90° from Figure 2A and highlights the positions of the neck α-helix (red) of Vik1MHD (left) and two different positions (red and blue) adopted by the neck α-helix of the N600K mutant form of Ncd (PDB accession:1N6M) (Yun et al., 2003). For the Ncd structures, only the α-carbons of residues found in the two motor domains have been superimposed. The α-carbons for the Vik1MHD, excluding the neck, were superimposed onto those of the Ncd motor domain. Atoms for the conserved glycine residue that allows neck rotation, Gly³⁷³ in Vik1 and Gly³⁴⁷ in Ncd, are shown as cyan and yellow spheres.

Figure 7. Models of the Kar3/Vik1 Motility Mechanism

The schematic drawings show two possible models for the interaction of the Kar3/Vik1 heterodimer with α/β-tubulin protofilaments during the motility cycle. The microtubule is oriented so that the minus end is on the left. In Model A, the coiled-coil (yellow) formed between the necks of Kar3 and Vik1 unwinds to allow both Kar3 and Vik1 to bind the same microtubule protofilament in a similar orientation, analogous to processive kinesins. In Model B, the coiled-coil between Kar3 and Vik1 does not unwind, keeping the globular domains of Kar3 and Vik1 close to each other. This imposes restraints on their interaction with the microtubule, such that the Kar3MD and Vik1MHD would have to interact with adjacent protofilaments and that Vik1 must bind α/β-tubulin in a different orientation than the motor domain of kinesins. In both models, the binding of the Kar3MD to the microtubule stimulates a conformational change in the motor that generates strain in the coiled-coil between Kar3 and Vik1. This strain produces a conformational change in the Vik1MHD that weakens Vik1’s interaction with the microtubule, which allows the large rotation of Kar3’s neck toward the minus end upon entry of ATP into the active site of Kar3 and complete disengagement of Vik1 from the microtubule. The direction of rotation of the coiled-coil and the timing of this event during the motile cycle is based upon cryo-electron microscopy studies of Ncd-decorated microtubules (Wendt et al., 2002; Endres et al., 2006).