A kinesin motor in a force-producing conformation

Abstract

Background: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states.

Results: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1.

Conclusions: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.

Figure 1

NcdT436S. A, T436S (blue) lies in the P-loop (green). ADP, purple; Mg²⁺, magenta sphere; water, green spheres. See also Additional file 1 Figure S1. B, Wild-type Ncd (PDB 1CZ7) [17] with T436 (blue).

Figure 2

NcdT436S nucleotide hydrolysis kinetics and motility. A, Mant-ADP release upon adding ATP (arrow). Assays performed in HEM100. NcdT436S (black, red curve fit); wild type (WT) (grey, pink curve fit). a.u., arbitrary units. B, Mant-ADP release upon adding excess microtubules (MTs, arrow), then ATP (arrow). Assays performed in HEM50. NcdT436S (black, red curve fit) released ADP with no additions, then, after adding microtubules, released most of the remaining bound ADP; the rest was released by adding ATP. By contrast, wild type (grey, pink curve fit) released about half the bound ADP with microtubules and the rest upon adding ATP. C, Basal and microtubule-stimulated ATPase activity in steady-state assays. NcdT436S (black, red curve fit); wild type (grey, pink curve fit). k_cat ± sem. D, In vitro motility. Leading-end microtubule gliding velocity ± sem on NcdT436S and wild type.

Figure 3

NcdT436S crystal structure. A, NcdT436S heads are oriented asymmetrically relative to the coiled-coil stalk. Chain A, cyan; chain B, green. B, Wild-type Ncd (PDB 1CZ7) [17] heads show two-fold symmetry around the axis of the stalk. Bottom, structures rotated 90°; the stalk extends out of the plane of the page highlighting the different orientation of the chain B head.

Figure 4

Salt bridges between the motor core and neck in NcdT436S.Distinct salt bridges between the neck and motor core of chain A (cyan) and B (green) stabilize the orientation of the heads with respect to the neck, as indicated. Dimer orientation the same as Figure 3A (top).

Figure 5

Docking of the NcdT436S C-terminus onto the motor domain. The C-terminal seven visible residues (NSCKMTK) after helix α6 (green) of chain B pack into a groove formed by the C-terminus of helix α4, N terminus of strand β8 of the central β-sheet, and N-terminus of the neck helix. The C-terminus of chain A (see Figure 6A) projects away from the motor domain and quickly becomes disordered.

Figure 6

NcdT436S vs KIF1A. A, NcdT436S chain A (cyan) and B (green) differ in C-terminus orientation (chain A, magenta; chain B, pink), helix α4 differs in length and the neck helices show distinct trajectories as they leave the motor domain to form the coiled-coil stalk. B, Helix α4 is longer by two turns in KIF1A-ADP (dark cyan) than KIF1A-AMP·PNP (dark green; C-terminus, brown). C, Helix α6 is oriented similarly in NcdT436S chain A and KIF1A-ADP. D, The C-terminus of NcdT436S chain B and KIF1A-ATP (KIF1A-AMP·PNP) are similarly oriented and helix α4 is similar in length. See also Additional file 1 Figure S2.