The Mechanochemical Cycle of Mammalian Kinesin-2 KIF3A/B under Load

Abstract

The response of motor proteins to external loads underlies their ability to work in teams and determines the net speed and directionality of cargo transport. The mammalian kinesin-2, KIF3A/B, is a heterotrimeric motor involved in intraflagellar transport and vesicle motility in neurons. Bidirectional cargo transport is known to result from the opposing activities of KIF3A/B and dynein bound to the same cargo, but the load-dependent properties of kinesin-2 are poorly understood. We used a feedback-controlled optical trap to probe the velocity, run length, and unbinding kinetics of mouse KIF3A/B under various loads and nucleotide conditions. The kinesin-2 motor velocity is less sensitive than kinesin-1 to external forces, but its processivity diminishes steeply with load, and the motor was observed occasionally to slip and reattach. Each motor domain was characterized by studying homodimeric constructs, and a global fit to the data resulted in a comprehensive pathway that quantifies the principal force-dependent kinetic transitions. The properties of the KIF3A/B heterodimer are intermediate between the two homodimers, and the distinct load-dependent behavior is attributable to the properties of the motor domains and not to the neck linkers or the coiled-coil stalk. We conclude that the force-dependent movement of KIF3A/B differs significantly from conventional kinesin-1. Against opposing dynein forces, KIF3A/B motors are predicted to rapidly unbind and rebind, resulting in qualitatively different transport behavior from kinesin-1.

Figure 1. KIF3A/B stepping against hindering load in an optical force clamp

(A) Recombinant kinesin constructs used in the study. KIF3A/B (blue label) consists of the full-length KIF3A (green) and KIF3B (red) sequences fused to a C-terminal His₆ tag (pink). Kinesin-1 (black label) is a truncated DmKHC construct (black) fused to the GFP sequence (orange) and a His₆ tag. Homodimeric mutants were generated by joining the KIF3A or KIF3B motor domains to the stalks of KIF3A/B or Kinesin-1. The splice site was the junction between the neck linker and stalk for the respective motors. Two additional mutants of 3A-KHC were also created: 3A-KHC_P>A replaces the KIF3A NL proline (P, bold) by alanine, and 3A-KHC_P>A,ΔDAL, which carries the identical mutation, together with a deletion of the three C-terminal neck-linker residues (DAL, underscored). (B) Representative records of single-molecule movement for KIF 3A/B (4 pN hindering load, 5 μM ATP) displaying forward steps of 8 nm (blue), backsteps of 8 nm (olive) and slips of variable distance (red).

Figure 2. Motor velocity and randomness as a function of load and ATP

(A, B) Force-velocity curves from force-clamp optical trapping experiments. (C, D) Velocity at varying ATP concentrations under zero load and 4 pN hindering load. (E) The randomness parameter, r, as a function of force at 2 mM ATP. (F) The randomness parameter, r, as a function of ATP at 4 pN hindering load. Data points and error bars (s.e.m.) indicate experimental velocities or randomness values. Solid curves were derived from a global fit to the data. (see Figure 3, Tables S1, S2, and Supplemental Experimental Procedures). Velocity and randomness results for KIF3A/A in 5 μM ATP are shown in Figures S2A, S2B. Comparisons between KIF3A/B and kinesin-1 randomness vs. force or ATP concentration are shown in Figures S2E and S2F.

Figure 3. Modeling the KIF3A/B mechanochemical cycle

(A) Processive stepping pathway for KIF3A/B. Transitions k_5A and k_5B (dark red arrows) are associated with backsteps. (B)Legend for the cycle in (A). KIF3A (green) and KIF3B (red) form the KIF3A/B dimer that moves on MTs (brown). (C) Table of fit parameters and standard errors of fit for the global fit of the kinetic model to the data of Figures 2, S2A and S2B. Assignments of the mechanochemical transitions that correspond to each rate constant in the pathway are indicated. A three state model (combining states 3 and 4) was sufficient to model most of the data; however, fitting the model to the randomness data required four states for head B. The data for head A were not sufficient to constrain parameter k_4A, so in the actual fit, states 3A and 4A were lumped, equivalent to assuming a very rapid transition [3A] → [4A]. The lower bound for this transition was estimated using FitSpace. Similar model fits were carried out for kinesin-1 and kinesin-2 mutants (Tables S1, S3).

Figure 4. Dependence of processivity on load and ATP

(A) Run length for KIF3 constructs as a function of force, together with kinesin-1 (in dark grey). Mean run lengths (± s.e. of fit) at each force were calculated from exponential fits to the run-length distribution. Solid curves represent fits to the expression L = L₀ exp (−F·δ/k_BT), where L_o is the run length extrapolated to zero load from hindering-load data and δ is the distance parameter. (B) KIF3 data from panel (A) rescaled, showing the differences among motor constructs. (C, D) ATP dependence of KIF3 run lengths under zero load and 4 pN hindering load. Values are mean ± s.e. of fit. Solid lines show fits over all ATP levels. (E) Table of parameters for fits to data in (A) and (B). L_obs is the unloaded run length obtained by video tracking, averaged for all ATP concentrations. Run length results for KIF3A/A in 5 μM ATP and for 6 pN load are displayed in Figures S2C and S2D.

Figure 5. Unbinding force measurements

(A) Unbinding force histograms for 3A-KHC, 3B-KHC and kinesin-1 (rows) in 1 mM ADP, 0.5 mM AMP-PNP, or no nucleotide (columns). Negative unbinding forces correspond to pulling kinesin toward the microtubule minus-end (the hindering load direction); positive forces are toward the plus-end (assisting load direction). Loading rates: 100 pN s⁻¹ for ADP; 10 pN s⁻¹ for AMP-PNP and no nucleotide (apyrase). Solid lines represent fits to the data under each curve for each pulling direction. Bins with few counts were excluded as well as data at low forces due to the possibility of missed events. (B) Fit parameters (± s.e. of fit), are shown for each construct and experimental condition, using the parameters indicated in Eq. (1). k₀ is the unloaded off-rate and δ is the characteristic distance parameter. For microtubule dissociation rates during processive stepping, see also Figure S4.

Figure 6. Effect of neck-linker length on force-dependent properties of KIF3

Data from chimeric constructs consisting of kinesin-2 heads fused to the kinesin-1 stalk (2 mM ATP). (A) Load dependence of run length for 3A-KHC constructs with different neck-linker domains, along with 3B-KHC, color-coded as in panel B. Values at each force are mean ± s.e. of fit to exponential run length distributions. Run lengths as a function of force were fit to exponential functions; parameters are given in Table S4.(B). Inset: Expanded view of run lengths at non-zero loads. (B) Load dependence of velocity, colored as shown (legend). Velocity data were fit using a 3-state model with a single force-dependent transition. Parameters are given in Table S3. A comparison between velocities and run lengths for homodimers with the native KIF3A/B stalk (KIF3A/A and KIF3B/B) and the kinesin-1 stalk (3A-KHC and 3B-KHC) is shown in Figure S5. The corresponding data for kinesin-1 with an extended neck linker are displayed in Figure S6 and Table S1