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

Abstract

As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."

Fig. 1.

Experimental design. (A) Schematic of experiment. Dimeric kinesin-1 with two heads (Gray) bound to a microtubule (Light Blue). Microtubules are stuck to a glass slide and observed using TIRF microscopy. If the kinesin neck (Gold) is tangent to the microtubule surface (Right), FRET between a donor (Blue) on the microtubule and an acceptor (Red) at the end of the neck will be high. If the kinesin neck is oriented radially (Left), FRET will be low. (B) Three-dimensional structure of one subunit of the kinesin dimer (21) oriented such that the microtubule binding surface (Green) would bind to the top of the microtubule in (C). Cy5.5 was attached to residues on the head and neck marked with red space-filled atoms. (C) EM reconstruction, shown end-on, of three protofilaments of a microtubule (43). Bound taxol is marked with blue space-filled atoms.

Fig. 2.

FRET between microtubules and kinesin. Each TIRF microscopy image shows long-wavelength (> 635 nm) fluorescent emission from microtubules immobilized on a coverslip under 532 nm (donor) excitation. (A) TAMRA-X-taxol microtubules decorated with kinesin with Cy5.5 on C45. Decoration density 0.1 kinesin heads per tubulin dimer. The emission in this image includes contributions from FRET, leak-through donor emission and direct acceptor excitation. (B) Undecorated TAMRA-X-taxol microtubules, showing the emission due to leak-through. (C) Unlabeled microtubules decorated with kinesin with Cy5.5 attached to C45, showing the emission due to direct excitation. Bar: 5 μm; intensity scale (Right, a.u.) applies to all three images. Microtubule fluorescence in (A) is significantly larger than the sum of intensities in (B) and (C), demonstrating FRET.

Fig. 3.

Comparison of FRET to positions on kinesin head with predictions based on EM reconstructions. Black: Predicted FRET; line width indicates the range of predictions from two different EM reconstructions. Gray: histograms of FRET measurements, one value per microtubule. FRET was from TAMRA-X-taxol to Cy5.5 attached at C45 (A; N = 72), A128C (B; N = 73), and S181C (C; N = 18). The prediction lies within ± 2 × S.E. of the measured value in (A) and (C); in (B) there is a possibly significant but still small (ΔE =  ∼ 0.14, corresponding to ∼11 Å) deviation, perhaps attributable to a small perturbation of the loop structure by the cysteine substitution at position 128.

Fig. 4.

The kinesin neck is oriented parallel to the microtubule surface. (A) Points: FRET efficiencies (mean ± 95%C.I.) to Cy5.5 attached at three different sites on the kinesin neck (K349C [N = 86], G364C [N = 118] and A378C [N = 92] are at 0, 23, and 43 Å from the base of the neck, respectively). Black Line: best fit neck orientation. Red Line: radial neck orientation. (B) End-on view of the top protofilament of a microtubule (Blue, β-tubulin; Pink, α-tubulin) and a bound kinesin dimer (Yellow, Green) with the neck oriented almost parallel to the microtubule surface according to the best fit of (A). The fit parameters, angle α and height h, are marked. The value of h (measured at K349C, four residues after the base of the neck) agrees with the height of the base of the neck (Green Dot) determined by EM reconstructions. (C) View from above of one protofilament of a microtubule and two bound kinesin dimers modeled with a surface-parallel neck (α =  ∼ 0°) in either the axial (θ = 0°) or tangent (θ = 90°) orientations (Red Arrows). Both orientations are consistent with the FRET data, but only the nonaxial orientation is reasonable due to structural constraints (see text).

Fig. 5.

Ruling out alternative explanations for neck orientation. (A) Effect of kinesin density on efficiency of FRET to Cy5.5 at the end of the neck (Blue, mean ± 95%C.I., N = 16 - 44), and of BCCP/HIS tag removal (Red, N = 76). Wide error bar at the lowest density results from low signal/noise. (B) Effect of E-hook removal by subtilisin digestion of microtubules (Red, N = 30 - 41) on FRET efficiency (mean ± 95% C.I.). Data for undigested microtubules (Black) is shown for comparison.