Kinesin is an evolutionarily fine-tuned molecular ratchet-and-pawl device of decisively locked direction

Abstract

Conventional kinesin is a dimeric motor protein that transports membranous organelles toward the plus-end of microtubules (MTs). Individual kinesin dimers show steadfast directionality and hundreds of consecutive steps, yet the detailed physical mechanism remains unclear. Here we compute free energies for the entire dimer-MT system for all possible interacting configurations by taking full account of molecular details. Employing merely first principles and several measured binding and barrier energies, the system-level analysis reveals insurmountable energy gaps between configurations, asymmetric ground state caused by mechanically lifted configurational degeneracy, and forbidden transitions ensuring coordination between both motor domains for alternating catalysis. This wealth of physical effects converts a kinesin dimer into a molecular ratchet-and-pawl device, which determinedly locks the dimer's movement into the MT plus-end and ensures consecutive steps in hand-over-hand gait. Under a certain range of extreme loads, however, the ratchet-and-pawl device becomes defective but not entirely abolished to allow consecutive back-steps. This study yielded quantitative evidence that kinesin's multiple molecular properties have been evolutionarily adapted to fine-tune the ratchet-and-pawl device so as to ensure the motor's distinguished performance.

FIGURE 1

Stepping barriers and configurational energies of conventional kinesin. Kinesin-MT binding configurations are illustrated in the insets. The motor heads are represented by large symbols filled in yellow color. The ATP-bound state of a head is indicated by label T, and the ADP-bound state by label D. Unlabeled heads are nucleotide-free. The neck linkers are shown by lines in blue color, and their zippered portions are shown by bold lines in red. The coiled coil dimerization domains are shown by spiral lines in cyan. The large symbols in dark and light gray represent α- and β-tubulin units of MT. (A) Lowest free-energy barriers for forward and backward stepping. The solid symbols are calculated results for integer numbers of zippered amino-acid residues, while the lines were drawn to guide the eye. The bias, i.e., barrier difference between forward and backward steps, is also shown. The measured values for the barriers are from Taniguchi et al. (14). (B) Computed energies for major kinesin-MT binding configurations as a function of hypothetically changing length of the linker peptide. The solid symbols are results for integer numbers of amino-acid residues in a linker peptide, and the lines were drawn to guide the eye. Conventional kinesin's effective linker length for double-headed bindings to MT is indicated by the shaded area. (C) Distortion of configurational hierarchy by opposing load. The shaded area indicates the measured stall forces from Visscher et al. (3), which are 5–8 pN, depending on values of ATP concentrations.

FIGURE 2

Walking behavior of kinesin dimers. (A) Illustration of kinesin's major mechanochemical cycle at low loads deduced from the configurational analysis (see text). The kinesin-MT system and the states of the motor heads are illustrated in the same way as in the insets of Fig. 1 B. At low loads, three dimer-MT binding states (I–III) are likely involved. The transition from state I to II is caused by ATP binding and linker zippering at a MT-bound head. MT binding and ADP release of the diffusing head causes transition from state II to III. Hydrolysis-initiated detachment of the rear head causes transition back to state I. (B–F) Prediction of the kinetic Monte Carlo simulation (solid lines) versus experimental data (solid symbols). (B) Typical trajectories of both heads. Initially at zero time the two heads are both bound to MT. After hydrolysis-initiated detachment, the diffusing head is allowed to bind MT again only at binding sites other than the one occupied by the standing head. Thus the head whose trajectory is shown by solid lines in red (black) binds MT only at positions indicated by red (black) dashed lines. A color mismatch between solid lines (head trajectories) and dashed lines (MT sites) indicates the diffusing state of a head. (C,D) Average velocity of the dimer as a function of ATP concentrations and opposing loads. The measured data are from Visscher et al. (3). (E,F) Temporal fluctuation of the dimer's walking steps as a function of ATP concentrations and loads. The measured data are from Schnitzer and Block (50) for panel E and Visscher et al. (3) for panel F. The overall mechanochemical coupling ratio; namely, average number of ATP molecules consumed per forward step is also shown in panel F.

FIGURE 3

Robustness of kinesin's ratchet-and-pawl. Lower boundary (n₁) and size (Δn = n₂ − n₁) of the ideal working regime for the ratchet-and-pawl mechanism in terms of effective linker length as a function of hypothetical changes in zippered length of neck linkers upon zippering (n_z), associated free-energy gain (U_z), and head-MT binding energies for a nucleotide-free or ATP-bound head (U_KM). (Both binding energies were assumed equal in obtaining the results shown by the figures.) For other kinesin-MT parameters, the same values as for Fig. 1 are used. Definitions of n₁ and n₂ are shown in Fig. 1 B. The n₁ and Δn values for conventional kinesin are indicated by the open areas.