What kinesin does at roadblocks: the coordination mechanism for molecular walking

Abstract

Competing models for the coordination of processive stepping in kinesin can be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide-induced detachment of kinesin monomers or dimers with and without the T93N roadblock using microflash photolysis combined with stopped flow. Control nucleotide-induced monomer (rK340) unbinding was 73.6 s(-1) for ATP and 40.5 s(-1) for ADP. Control ADP-induced dimer (rK430) unbinding was 18.6 s(-1). Added 20 mM Pi slowed both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers is prevented and ATP-induced trail head unbinding was then 42 s(-1). This is less than two-fold slower than the stepping rate of unimpeded rK430 dimers (50-70 s(-1)), indicating that during walking, lead head attachment induces at most only a slight (less than two-fold) acceleration of trail head detachment. As we discuss, this implies a coordination model having very fast (>2000 s(-1)) ATP-induced attachment of the lead head, followed by slower, strain-sensitive ADP release from the lead head.

Figure 1

Nucleotide-induced kinesin dissociation from MTs. (A, B) Light-scattering traces obtained by flash photolytic release of nucleotide. (A) rK340 monomers (0.8 μM) were dissociated from MT (0.4 μM) by flash photolytic release of 60.0 μM ATP or 66.3 μM ADP (as indicated). Single exponential fits to the traces (solid lines superimposed) gave k_obs values of 21.2 and 10.6 s⁻¹ for the ATP and ADP cases, respectively. (B) Similar traces obtained with rK430 dimers at 62.1 μM ATP (k_obs=7.7 s⁻¹) and 65.6 μM ADP (k_obs=4.8 s⁻¹). (C, D) Light-scattering traces obtained by stopped flow. (C) rK340 monomers (0.8 μM) were dissociated from MT (0.4 μM) by 750 μM ATP (k_obs=51.6 s⁻¹) or ADP (k_obs=26.7 s⁻¹). (D) rK430 dimers (0.8 μM) were dissociated from MT (0.4 μM) by 750 μM ATP (k_obs=33.4 s⁻¹) or ADP (k_obs=29.3 s⁻¹). (E, F) Combined flash-photolysis data and stopped-flow data. (E) Unbinding rate constants for ATP- (filled symbols) or ADP (empty symbols)-induced unbinding of rK340 monomers (0.8 μM) from MT (0.4 μM). (F) Unbinding rate constants for ATP- (filled symbols) or ADP (empty symbols)-induced unbinding of rK430 dimers (0.8 μM) from MT (0.4 μM). The flash-photolysis data (on the left of the vertical dashed line in (E) and (F)) were corrected for inhibition by caged nucleotide as described in Materials and methods. Hyperbolic fits to the corrected rate constants are shown as solid lines. Values obtained for the parameters of the hyperbolic fits are shown in Table I. In all cases, the concentrations given are mixing cell concentrations.

Figure 2

Characterisation of T93N. (A) MT pelleting experiment showing the irreversibility of the binding of T93N to MT. Left of markers: controls. Supernatants and pellets following centrifugation of wild-type kinesin alone, MT alone and T93N alone in the absence of ATP. Right of markers: ATP-induced unbinding. The first three lanes show the T93N experiment. A complex of MT and T93N was formed and centrifuged to produce a pellet P and a supernatant S1. The pellet P was resuspended in an ATP-containing solution and recentrifuged. The resulting supernatant S2 contained no detectable T93N, indicating that its binding to MT is effectively irreversible. The last three lanes show an rK340 positive control. In this case the ATP releases a large fraction of the rK340 into the supernatant S2. (B) Influence of the concentration of T93N on the rate of MT-activated ATPase of rK340 (open squares) and rK430 (filled squares). (C) Inhibition by T93N of MT gliding motility on a surface of rK430 GST. MTs were preloaded with T93N at the indicated occupancy and then introduced into the assay chamber. The maximum velocity (no T93N) under these conditions was 382 nm s⁻¹. Both ATPase and motility assay data were obtained in the same buffer and at the same temperature as the transient data.

Figure 3

Effect of T93N on ATP-induced kinesin–MT unbinding. In total, 0.2 μM kinesin dimer was dissociated from 0.4 μM MT by flash photolytic release of ATP or by stopped-flow mixing with ATP. MTs were prereacted with T93N at the indicated occupancy. (A) Example of light-scattering traces obtained by microflash photolysis at 0, 0.2, 0.4 and 0.6 μM T93N. The superimposed solid lines are single exponential fits to the data (see Materials and methods). (B) Example of light-scattering traces obtained by stopped flow at 0.75 mM ATP to define k_max. The data are fitted to single exponentials. (C) Plots of the inverse of the observed rate constants (k_obs⁻¹, the residence time) against T93N concentration (data from stopped flow). (D) ATP dependence of the unbinding rate of 0.2 μM rK340 monomers in the absence of T93N (open circles) or in the presence of 0.4 μM T93N (filled circles). Crowding of rK340 in the absence of T93N also has no effect: filled squares show an unbinding of 0.8 μM rK340.

Figure 4

Proposed kinetic scheme for monomers. Nucleotide binding is followed by an OPEN-to-CLOSED conformational change (see text). MT binding and unbinding are via an M.K^TRAPPED.ADP intermediate. Rate constants for nucleotide binding and dissociation are consistent with the literature values (e.g. Ma and Taylor, 1997a, 1997b; Gilbert et al, 1998). The rate constant for complex formation of K^TRAPPED.ADP and M is close to the diffusion limit (Hackney, 1995). Values for the other rate constants are estimates as indicated by the tildes (∼), and were made by simulating the scheme in Berkeley Madonna 8.1b6 and in KSIM (Neil Millar) using the current data (ADP-induced dissociation of M.K∼40 s⁻¹; ATP-induced dissociation of M.K∼70 s⁻¹; K_m for ATP∼75 μM; K_d for ADP binding to M.K∼120 μM) as constraints, and the following further constraints: K_d for K.ADP-MT∼20–30 μM (Crevel et al, 1996); MT-activated transient release of ADP from K.ADP∼100 s⁻¹ (Rogers et al, 2001).

Figure 5

Logical gating scheme for dimers. The scheme shown refers to molecular walking at low external load, and corresponds to the chemical kinetic release of ADP from the lead head induced by the binding of ATP to the trail head. Following ATP binding to the trail head, tight attachment of the lead head to its next site occurs rapidly (>2000 s⁻¹), but subsequent MT-activated ADP release from the lead head is inhibited by distortions due to minus endwards tension (black chevron). Relief of this strain by release of the trail head from the MT then allows ADP release from the lead head. The mechanochemical interdependencies of the two heads are like logic (IF...THEN DO) gates, as shown.

Figure 6

Scheme I