Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family

Abstract

Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin--the transport motor kinesin-1--is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor's structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements.

Keywords: cancer; macromolecular assemblies; mitosis; molecular motors.

Fig. 1.

The ATP hydrolysis transition state: MD tilting, L5 uncurling, and CNB destabilization. (A) MT-bound K5 MD in the ADP.AlFx state (light blue surface: 1.1σ contour) with gold difference maps superimposed (blue: 2.3σ; pink: 2.2σ; red: 2.2σ contour). In all figures, the MT plus end is towards the top. (B) Pseudo-atomic models of K5 MD (color-coded as in the key) bound to an αβ-tubulin dimer (green ribbon) docked into the reconstruction (contour as in A; mesh: 3.3σ contour, ∼1/3 of the volume). Residues to which gold clusters were covalently attached and the nucleotide analogue are shown in space-filling representation. NL conformers clustered into docked (red) and disconnected (orange) conformations are indicated (SI Materials and Methods). (C and D) View towards the nucleotide-binding pocket of the AMPPNP (15) (C: coral surface: 1σ contour, mesh: 3σ contour, to depict the same molecular volumes as in B) and ADP.AlFx (D: contoured as in B). The blue arrow indicates the absence of contact between helix α0 and switch I in the ADP.AlFx state, which is present in the AMPPNP state. (E) Comparison of K5 MD models of ADP.AlFx (colored as in the key) and AMPPNP states [coral (15)], superimposed on helices α4. ATP hydrolysis triggers a tilt of the MD towards the MT lattice (colored arrow). L5 uncurls from its arch-like conformation upon ATP hydrolysis (comparison based on superimposed helices α2). Displacements of helix α3/switch I and helix α0 are caused by MD tilting.

Fig. 2.

The ADP MT weak-binding state. (A) ADP state (1.5σ contour, the same volume as in Fig. 1A) with gold density difference maps superimposed (blue: 1.5σ; pink: 2.5σ; red: 1.9σ contours). (B) Pseudo-atomic model of K5 MD bound to an αβ-tubulin dimer (surface contoured as in A; mesh 3σ contour 1/3 of the surface volume). The red arrow indicates the absence of EM density accommodating the N-terminal part of the NL and the C-terminal part of helix α6 to which it is attached. (C and D) Comparison of K5 MD pseudoatomic models of the ADP.AlFx (C) and ADP (D) states; surfaces and meshes are contoured as in Fig. 1C (C, ADP.AlFx) and Fig. 1B (D, ADP). The yellow arrow indicates the lack of density for switch II that therefore is absent in our ADP model. (E) The ADP MT weakly bound state is rotated clockwise with respect to the MT axis compared with the ADP.AlFx state (color-coded arrows). The surface is contoured at 2σ; for clarity, only some of the secondary structural elements are shown.

Fig. 3.

Conformational changes associated with ADP release. (A and B) K5 MD pseudo-atomic models of the ADP (light purple) and rigor states are superimposed on helices α4 and docked into the rigor reconstruction (15); surface: 0.94σ contour; mesh: 3σ contour. (A) The position of the gold density (blue surface, 1.4σ contour) attached to the N terminus via A9C at ∼23 Å or approximately seven amino acids away from the first residue in our rigor model (G16) confirms our previous assignment of the N terminus in the rigor reconstruction (15). The color-coded arrow indicates the direction of the MD tilt around the helix α4 and towards the MT lattice upon ADP release. (B) Upon ADP release, L5 moves towards the empty nucleotide-binding pocket, based on superimposition of helices α2. The black arrowhead and the blue arrow indicate a connection between L5 and switch I (SI) and between L5 and helix α0, respectively. Helix α3/switch I, helix α4, and helix α0 of the ADP model also are shown for comparison.

Fig. 4.

Effect of deletion of the K5 N terminus on kinetic parameters and motility. (A) The jagged curves represent the fluorescence transient observed by mixing TMR-labelled NT-Intact MT (Left) and TMR-labelled NT-Deleted:MT (Right) complex with 800 μM ATP or ADP, and the smooth curves are fit to a double (red) or single (blue) exponential rate equation. (Insets) Plots of rate constant versus [ATP] or [ADP]. (B) Individual MT gliding traces for NT-Intact (blue) and NT-Deleted (red) constructs. Each set of traces (n = 17 and 22, respectively) was averaged together pointwise to extract average behavior [bold traces: blue, 25.7 nm/s; red, 17.4 nm/s; error bars (light-colored zones) indicate SEM; **P < 0.001]. Individual frames were 85 ms apart (SI Materials and Methods).

Fig. 5.

Molecular adaptations of kinesin motors: N terminus/NL conformational coupling during the mechanochemical cycle. The central schematic illustrates the MT-based ATPase cycle: ATP hydrolysis (step A), Pi release/MT detachment and MT re-binding (step B), ADP release (step C), and ATP binding (step D). In the corners of the figure, the major nucleotide-dependent conformational changes in the K5 N terminus and NL are depicted. The large seesawing motion of the MD at steps B and D is shown by rotation of the K5 MD on the αβ-tubulin dimer. The proposed route of structural communication between helix α0 proximal to the active site via strand β1 is shown. Stability of the N terminus/NL is depicted as a solid line; flexibility is indicated by a dotted line. CNB formation is indicated by a black square. The outer cycle depicts the conformational changes in the K1 MD (24, 30). In contrast to K5, however, both the K1 N terminus and NL are highly flexible in both the ADP and rigor states. The exact route of structural communication between the K1 helix α0 and N terminus has not been described.