Conformation of the switch loops and switch II helix near the active site. (A) Overview of the nucleotide pocket face of the motor domain, with labeled components. (B)–(D) close-up views of the nucleotide pocket in our ADP, no-nucleotide, and ADP•Al•F_x maps, respectively, with crystal structure models of K349 superposed. In (D), our phosphate tube model for the switch loop residues (Green) has been substituted for the crystallized K349 switch loops. Location of the “switch pocket” (Panel I ) is indicated. Movie S6 depicts the hybrid atomic model for our K349 construct, as seen in this panel, juxtaposed with our ADP•Al•F_x map. (E) Network of stabilizing interactions formed following formation of the phosphate tube and occupation of the switch pocket by the switch II helix extension. Side chains of residues E250 and Ile 254 are rendered as red van der Waals spheres, while conserved switch loop residues 201–205 (from switch I) and 233–236 (from switch II) are rendered as ball-and-stick diagrams. Vantage point is the same as (A)–(D). Movie S5 illustrates this arrangement in 3D. (F) Molecular surface formed by the switch loops in crystal structure 1MKJ. Surface is rendered in the CPK coloring scheme; view is from the interior of the microtubule, looking out at the nucleotide pocket. (G) Molecular surface formed by the phosphate tube conformation of the switch loops, showing the “switch pocket” complementary to E250 and Ile 254 from the switch II helix extension. The entry point into the phosphate tube, which leads to the γ-phosphate, is visible as a fenestration in the middle of the switch pocket.

Predicted interactions of modeled switch loop conformations in no-nucleotide and ADP•Al•F_x maps. Similar results were obtained for the ADP and AMPPNP maps (see Fig. S5). The positions of residues R203 (switch I; main chain atoms only, Orange Sphere depiction), A233 (switch II; Red Sphere depiction), and Ile 254 from the switch II helix extension (Dashed Outline, Red Sphere depiction) are indicated. Beta strands connected to the switch I and switch II loops, running through the motor domain’s central β-sheet, are rendered as ribbon diagrams, and the nucleotide is rendered as a ball-and-stick diagram. A thin cross section of the density map isosurfaces, aligned with the depicted β-strands and running through the predicted position of R203 and Ile 254, is also shown, as is density for the switch II helix. (A) Analysis of the no-nucleotide state, predicting complementary packing between the side chain of Ile 254 from the switch II helix extension and residues R203, A233 from the fitted K349 crystal structure model. Relative orientation of the catalytic core is indicated by the blue bars. (B) Substituting the phosphate tube loop conformations into the fitted K349 structure from (A) generates a hydrophobic cavity between Ile 254 and the switch pocket. Dashed line indicates rotation of the switch II helix required to occupy the cavity. (C) Analysis of the ADP•Al•F_x map predicts steric interference between Ile 254 and residues R203 and A233 from the fitted K349 structure. The black and white striped area indicates the predicted region of steric overlap. Dashed line indicates rotation of the switch II helix required to relieve the overlap. (D) Modeling of the phosphate tube loop conformations in the ADP•Al•F_x-bound map relieves the steric overlap found in the (C).

Summary of our atomic models describing microtubule-activated cargo displacement and catalysis by the kinesin motor domain. (A) Close packing between the switch loops and the switch II helix extension prevents leftward tilting and premature docking of the neck linker. The approximate path of the “nucleotide-ejecting” conformation of the switch I loop, inferred from cryoEM density (14), is indicated by the orange dashed line. Bulky hydrophobic residues, which we identify as the fulcrum of seesaw movement by the motor domain, are depicted by blue spheres. (B) Formation of the phosphate tube/switch pocket, caused by retraction of the switch loops toward the nucleotide pocket. In the absence of the microtubule-stabilized switch II helix extension, this retracted switch loop conformation would not be fully stabilized and is expected to rapidly revert to conformation(s) seen in kinesin crystal structures. The state depicted here is unlikely to be highly populated and is presented here to highlight the features that are caused by retraction of the switch loops. (C). Leftward tilting of the catalytic core domain leads to occupation of the switch pocket by residues E250 and Ile 254. This arrangement stabilizes the phosphate tube conformation of the switch loops, triggering catalysis and simultaneously triggering a power stroke by the neck linker (Magenta) on the opposite side of the catalytic core. In conventional kinesins, the cargo (and/or dimer partner) is attached to the magenta helix at the end of the neck linker and so is translocated toward the plus end of the microtubule in this step. The axis of rotation defined by our molecular fits of the K349 motor domain (no-nucleotide to ADP•Al•F_x transition) is depicted by bulls-eye symbol where the axis runs through the fulcrum residue F84 (see Fig. S3).

Cartoon depiction of microtubule activation. (A) Disabled mechanism of kinesin in the absence of microtubules represented by crystallized conformations of the conventional motor domain. When detached from the microtubule, kinesin was previously shown to fluctuate between these states (12). Docking of the neck linker, to which cargo is attached, accompanies movement of the central core domain (Large Blue Rectangular Shape) relative to the switch II helix (Red Elongated Rectangle). Movement of the core domain is coupled to neck linker docking, due to a collision between α6 (to which the neck linker is connected) and the switch II helix (Left Cartoon). This collision is indicated by the dashed outline at the end of α6. The red dashed line depicts a ∼15–20 residue segment (L11) that connects the switch II loop to the switch II helix but is disordered in crystal structures. Circles labeled R and A depict residues R203 and A233. (B) Cartoon depiction of the states presented in Fig. 3. The N terminus of the switch II helix (Left Side) is extended by several turns, stabilized by a microtubule contact (represented by the Small Red Circle embedded within the microtubule surface). In the stroke state of the motor (state 1), this extension props the seesaw to the right, forcing the neck linker to remain in the disordered, stroke state. Following ATP binding, the switch loops retract toward the γ-phosphate, causing the seesaw to tilt to the left, accompanied by docking of the neck linker, which constitutes a power stroke that would translate the cargo of conventional kinesin toward the microtubule plus end (state 3). Residue I254 in the switch II helix extension is depicted as the red circle labeled I.