Neck linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors

Abstract

Defining the mechanical and biochemical determinates of kinesin processivity is important for understanding how diverse kinesins are tuned for specific cellular functions. Because transmission of mechanical forces through the 14-18 amino acid neck linker domain underlies coordinated stepping, we investigated the role of neck linker length, charge, and structure in kinesin-1 and kinesin-2 motor behavior. For optimum comparison with kinesin-1, the KIF3A head and neck linker of kinesin-2 were fused to the kinesin-1 neck coil and rod. Extending the 14-residue kinesin-1 neck linker reduced processivity, and shortening the 17-residue kinesin-2 neck linker enhanced processivity. When a proline in the kinesin-2 neck linker was replaced, kinesin-1 and kinesin-2 run lengths scaled identically with neck linker length, despite moving at different speeds. In low-ionic-strength buffer, charge had a dominant effect on motor processivity, which resolves ongoing controversy regarding the effect of neck linker length on kinesin processivity. From stochastic simulations, the results are best explained by neck linker extension slowing strain-dependent detachment of the rear head along with diminishing strain-dependent inhibition of ATP binding. These results help delineate how interhead strain maximizes stepping and suggest that less processive kinesins are tuned to coordinate with other motors differently than the maximally processive kinesin-1.

Figure 1. Kin1 Run Lengths and Design of Kin1 and Kin2 Constructs

(A) Run length of control Kin1 from TIRF assay. Data were fit to a single exponential. (B) Run lengths of different Kin1 constructs as a function of their neck linker (NL) length. Error bars represent the standard error from exponential fits. (C) Amino acid sequence of the kinesin-1 (KHC) and kinesin-2 (KIF3A) neck linkers with the adjacent α6 (last helix in the head domain) and α7 (neck coiled-coil domain). The Kin1 construct includes the entire DmKHC sequence up to residue 559 (in the break between Coil1 and Coil2 of the rod domain [19]) followed by a C-terminal GFP and hexaHis tag. The Kin2 construct includes the KIF3A head and 17 amino acid neck linker domain ending at Leu₃₅₉ (red sequence) fused to the DmKHC neck-coil and rod, starting at Ala₃₄₅, the first residue in the neck-coil domain (blue sequence). The cartoon shows the structures of the Kin1 and Kin2 constructs. The neck linker sequences for all constructs used in this study are given in Table S1 of Supplementary Data.

Figure 2. Kin1 and Kin2 Run Lengths Scale with Neck Linker Length

(A) Force-extension curves of kinesin-1 and kinesin-2 neck linkers from Molecular Dynamics simulations. Solid lines are predictions of worm-like chain model for 14 and 17 residue polypeptide showing good fit for kinesin-1 (14 residues) and poor fit to kinesin-2 (17 residues). Data are replotted from Ref. [8]. (B) Predicted force-extension curve when the kinked proline in kinesin-2 is replaced with an alanine and the last three amino acids are deleted (Kin2_{PA_ΔDAL}), compared to kinesin-1. (C) Comparison of run lengths for Kin2 and Kin1 constructs having identical neck linker lengths (# of amino acids). (D) Run lengths following substitution of the cis proline in the Kin2 neck linker with alanine (Kin2_PA). Kin2_PA constructs containing 14, 15, and 17 amino acid neck linkers are Kin2_{PA_ΔDAL}, Kin2_{PA_ΔDA}, and Kin2_PA, respectively. Curve for control Kin2 motors is shifted 1.5 amino acids to the left to account for the cis proline. All run length and velocity values are given in Table S2 of Supplementary Data.

Figure 3. Neck Linker Positive Charge and Low Ionic Strength Enhance Processivity

(A) Comparison of Kin1 run length on the charge of the neck linker insert showing that while negative charge does not diminish processivity, adding positive charge does enhance processivity. Experiments were carried out in 80 mM PIPES buffer. (B) Effect of buffer ionic strength on control Kin1 and Kin1_+KAL run lengths, showing that in 12 mM PIPES buffer the diminished processivity due to the longer neck linker domain is compensated for by enhanced electrostatic interactions due to the added positive charge in the neck linker domain.

Figure 4. Modeling the Kinesin Chemomechanical Cycle

(A) Model for the kinesin chemomechanical cycle used to interpret the neck linker extension results. This framework is similar to a previous model [6], with the difference that motor unbinding from State 3 is combined into one rate constant k_{unbind_2H} for simplicity. Kinetic parameters are discussed in Supplementary Data and listed in Table S3. (B) Experimental Kin1 run length and velocity results plotted as a function of neck linker length. (C) Modeled run length and velocity from stochastic simulations of the model presented in 4A, using rate constant parameters given in Table S3. In these simulations, k_{on_ATP_2H} was set to 0.2 μM⁻¹s⁻¹ (10-fold above the best estimate from the literature [2]) and both k_{detach_φ} and k_{detach_T} were varied from 2000 s⁻¹ down to 20 s⁻¹ to model the effect of reduced strain on the trailing head due to extending the neck linker domain. Hence, for the model to account for the experimental results, extending the neck linker needs to alter two strain dependent mechanisms – detachment of the trailing head and ATP binding to the leading head. Additional simulation results are given in Figure S4.