Direct measurements of kinesin torsional properties reveal flexible domains and occasional stalk reversals during stepping

Abstract

Kinesin is a homodimeric motor with two catalytic heads joined to a stalk via short neck linkers (NLs). We measured the torsional properties of single recombinant molecules by tracking the thermal angular motions of fluorescently labeled beads bound to the C terminus of the stalk. When kinesin heads were immobilized on microtubules (MTs) under varied nucleotide conditions, we observed bounded or unbounded angular diffusion, depending on whether one or both heads were attached to the MT. Free rotation implies that NLs act as swivels. From data on constrained diffusion, we conclude that the coiled-coil stalk domains are Ϸ30-fold stiffer than its flexible ''hinge'' regions. Surprisingly, while tracking processive kinesin motion at low ATP concentrations, we observed occasional abrupt reversals in the directional orientations of the stalk. Our results impose constraints on kinesin walking models and suggest a role for rotational freedom in cargo transport.microtubule ͉ molecular motor ͉ rotation ͉ single molecule ͉ yaw with one degree accuracy C onventional kinesin (kinesin-1) is a motor protein that transports cargo along microtubules (MTs) in cells, converting the chemical energy of nucleotide hydrolysis into mechanical work (1). The structure of kinesin consists of twin catalytic motor domains (heads) that bind to the MT substrate with nucleotidedependent affinity, joined by neck linkers (NLs) to the N terminus of an extended, ␣-helical coiled-coil (CC) stalk (Fig. 1A). Considerable recent progress has been made in understanding the mechanochemistry of the kinesin motor domains, which enable individual molecules to move processively, undertaking hundreds of successive 8-nm steps while hydrolyzing one ATP molecule per step (2-4). However, little is known about the structure of the stalk or its role in the currently favored model for kinesin movement, the so-called ''asymmetric hand-overhand'' stepping mechanism (5-7). In this mechanism, the heads of kinesin advance in strict alternation, exchanging leading, and trailing roles at each step, analogous to a person walking (1). An asymmetric mechanism requires that the ''left'' and ''right'' steps be slightly different; however, in contrast with a symmetric hand-over-hand walk, it does not require continuous rotation of the kinesin stalk. Consistent with this model, a landmark experiment by the Gelles group (8) that tracked the angular motions of MTs driven by single kinesin motors affixed to a surface did not find evidence for large-scale rotations.Although no net rotations of the kinesin stalk are expected during asymmetric hand-over-hand motion, significant momentary angular changes are nevertheless possible. If kinesin were a nearly rigid molecule, Ϯ180°reorientations of the stalk would be anticipated whenever the two heads exchanged positions during stepping (1, 9). It has been speculated that flexible elements within the kinesin structure may relieve any such momentary torsional strain, facilitating forward motion. Although previous single-molecule studies foun...

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V

“…This myosin molecule differs from another motor protein, kinesin which has been suggested to either not rotate its stalk or rotate it 180° (28).…”

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry

“…If interactions with the E-hook prevent this neck rotation as the trailing head steps forward, kinesin would be forced to adopt an asymmetric mechanism in which the three-dimensional structures of the kinesin-microtubule complex differ in even-and odd-numbered steps, and the two heads alternately step past the neck on its left and right sides (5, 7). Asymmetry is well established experimentally: All or nearly all steps result in zero neck rotation (5,36). Furthermore, the structural differences between alternate steps could result in different stepping kinetics in alternate steps, and this is in fact observedunder applied force, homodimeric kinesin constructs "limp," meaning that the enzyme has different dwell times following even-and odd-numbered steps (4,6).…”

The mechanical properties of kinesin-1: a holistic approach

“…It is sometimes called the swivel and is a section of the polypeptide chains that is thought to allow both rotational and flexional freedom [29,39]. However, a recent study [40] suggests that there may be a section in the middle of the KHCs prone to form an α-helix and a coiled coil in the dimer, leading to enhanced rigidity.…”

“…During the motility cycle, in the state when only one head is without a nucleotide and is attached to a microtubule, the neck linker acts as a flexible connector. In this state, it is able to swivel and allows the attached stalk and cargo to rotate freely, as it is a single polypeptide chain possessing single carbon bonds [10,29]. On ATP binding, the neck linker docks rigidly to the rest of the motor head domain forcing the connection point of head to stalk forward by ∼2.7 nm [30].…”

“…Artificial substitution of the sequence does not affect maximal motile velocity or force generation, although coiled coil formation is important [31], suggesting this sequence conservation might be for regulation purposes rather than motility [34]. Although made up of two α-helices (∼35 amino acids in length [29]) in a rigid coiled coil structure, it is thought that this region could allow slippage [35], sliding [36] or even partial unravelling, as the amino-acid sequence contains non-ideal residues for the formation of a coiled-coil structure at its hydrophobic centre [37]. However, engineered cross-linking between the coils has shown that unravelling is not necessary for normal processive motion to take place [38].…”