EPR spectroscopy shows a microtubule-dependent conformational change in the kinesin switch 1 domain

Abstract

We have used site-directed spin-labeling and electron paramagnetic resonance spectroscopy to monitor a conformational change at the nucleotide site of kinesin. Cys-lite kinesin (K349 monomer) with the mutation S188C was spin labeled with MSL or MTSL. This residue is at the junction between the switch 1 region (which is a structure known to be sensitive to bound nucleotide in the G-proteins) and the alpha3-helix, adjacent to the nucleotide site. The spectra showed two or more components of mobility, which were independent of nucleotide in the absence of microtubules (MTs). The spectra of both labels showed a change of mobility upon binding to MTs. A more mobile spectral component became enhanced for all triphosphate analogs examined, AMPPNP, ADP.AlFx, or ADP.BeFx, in the presence of MTs, although the magnitude of the new component and the degree of mobility varied with nucleotide analog. The ADP state showed a much-reduced spectral change with a small shift to the more immobilized component in the presence of MTs. For kinesin.ADP.MT, a van't Hoff plot gave DeltaH degrees = -96 kJ/mol implying that the conformational change was extensive. We conclude there is a conformational change in the switch 1-alpha3-helix domain when kinesin binds to MTs.

FIGURE 1

Ribbon diagram of S188C kinesin. Human kinesin (Kull et al., 1996) with Cys-188 (yellow) at the junction of the α3-helix (cyan, aa 176–189) with the switch 1 region (magenta, aa 190–213). The side chain of Ser-188 projects out into the aqueous environment in the crystal structure. The nucleotide-binding domain contains the P-loop (blue), switch 1, and switch 2 (green) homologous to myosin-family motors and the G-proteins. The β5-ribbon, aa 205–216, is shown in orange.

FIGURE 2

EPR spectra of MSL-labeled KS188C taken at 20°C in the presence and absence of microtubules. The horizontal axis is magnetic field (width = 12.7 mT), the vertical axis is the derivative of absorption. The top three spectra are in the presence of 5 mM ATP analog. The bottom spectra are in the presence of ADP. Locations A–C in the top three spectra, and locations D and E in the ADP spectrum, are discussed in the text. All spectra (Figs. 2–5) are normalized with respect to the central peak height to enhance the visual display of spectral differences in the low-field region.

FIGURE 3

EPR spectra of MSL-KS188C taken at 5°C. The top three spectra are in the presence of ATP analogs. The bottom spectrum is in the presence of ADP.

FIGURE 4

EPR spectra of MTSL-KS188C taken at 20°C. The top three spectra are in the presence of ATP analogs. The bottom spectrum is in the presence of ADP.

FIGURE 5

EPR spectra of MTSL-KS188C taken at 5°C. The top three spectra are in the presence of ATP analogs. The bottom spectrum is in the presence of ADP.

FIGURE 6

(Top) EPR spectra of MT•MSL-KS188C•ADP at 2°C (solid line) and MSL-KS188C•ADP at 30°C (dashed line), taken as the immobile and mobile components of the spectra of MT•MSL-KS188C•ADP as a function of temperature. (Middle) Spectrum of MT•MSL-KS188C•ADP at 15°C (dashed line) and the fit (solid line) as a linear combination of the spectra in top panel. (Bottom) Difference spectrum for spectra in middle panel.

FIGURE 7

Plot of [immobile fraction]/[mobile fraction] (spectra of Fig. 6, top) versus 1/T for MSL-labeled MT•KS188C•ADP as a function of temperature. The spectral components representing the mobile and immobile probes were obtained as described in the Methods. The slope of the least-squares linear fit gives ΔH⁰.

FIGURE 8

An overlay of structures (Cα backbone) of representative kinesin-family motors is shown. The α3-helix is at the top; the contiguous loop 9 is at the bottom. Loop 9 is part of the switch 1 region. Color coding: kinesin aa 181–197, gray (Kull et al., 1996); ncd aa 530–546, blue (Sablin et al., 1996); kif1a aa 194–210, cyan; Kar3 aa 576–592, magenta (Yun et al., 2001); and Kar3-R598A (Yun et al., 2001), red. Kinesin Ser-188 and homologous residues (ncd Met-537, kif1a K201, Kar3 Lys-583) are shown as color-coded Cα space-filling spheres. For Kar3•ADP with the mutation R598A (red), the crystal structure becomes disordered one amino acid to the α3-helix side of the Lys-583 position. Loop 9 is disordered and the x-ray structure terminates.