Strain through the neck linker ensures processive runs: a DNA-kinesin hybrid nanomachine study

Abstract

The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains elusive. Here we created a hybrid nanomachine (DNA-kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA-kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C-terminal or Mid position) and sub-nanometer changes in the distance between the two heads on motility. Our results show that although the native structure of kinesin is not essential for processive movement, it is the most efficient. Furthermore, forward bias by the power stroke of the neck linker, a 13-amino-acid chain positioned at the C-terminus of the head, and internal strain applied to the rear of the head through the neck linker are crucial for the processive movement. Results also show that the internal strain coordinates both heads to prevent simultaneous detachment from the microtubules. Thus, the inter-head coordination through the neck linker facilitates long-distance walking.

Figure 1

Structure of DNA-kinesin. (A) A Cys residue was introduced to the surface of the kinesin Cys-Light-Mutant (CLM), after which a fluorescently labeled ssDNA was attached. By hybridizing the two DNA-kinesin monomers, we obtained ‘parallel' or ‘anti-parallel' DNA-kinesin. (B) 10% poly-acrylamide gel electrophoresis (PAGE). S-Cy3, 20 bp Cy3-labeled sense oligo nucleotide; AS-Cy5, 20 bp Cy5-labeled antisense nucleotide; M, Marker. Digestion of DNA-kinesin with restriction enzyme (KpnI) showed that the DNA was correctly hybridized (right lane). DNA was labeled at position 337 (see Figure 3A). (C) Gel filtration column experiments using a wild-type dimer (black line, K490CLM 215), DNA-kinesin heterodimer (red line, 20 bp S-Cy3+20 bp AS-Cy5), DNA-kinesin monomer (blue line, 20 bp AS-Cy5), wild-type monomer (green line, K336CLM 215). Note: we obtained similar results using 6 bp constructs (data not shown).

Figure 2

DNA-kinesin can move processively at the single molecule level. (A) Native kinesin coiled-coil was replaced with duplex DNA. DNA was labeled at position 342 (see Figure 2B). On hybridization, a FRET signal was observed. (B) Kymograph obtained by green laser (514 nm) excitation. Owing to the high FRET condition, motile spots appeared only in the Cy5 channel (see Supplementary Figure S1 for details). (C) Velocity of the DNA-kinesin (red circle) followed Michaelis–Menten kinetics, indicating the movement was ATP hydrolysis dependent. However, the V_max (235 nm/s) was slower than that of wild type (K490CLM 416-Qdot655; 525 nm/s, blue squares). Inset: velocity distribution of DNA-kinesin at 1 mM ATP. (D) Run length (130 nm) was shorter than that of wild type (1300 nm). See main text for details.

Figure 3

DNA length dependence of DNA-kinesin movement. (A) Structure of anti-parallel DNA-kinesin. The overall structure is similar to that of the neck linker-extended kinesin mutants. In addition, several connect positions were feasible in DNA-kinesin (see Figure 6 for details). This is in sharp contrast to a protein-only base mutant, in which only N- or C-terminal connections can be achieved. (Inset) DNA-kinesin connected at position 337, which is at the end of the neck linker, is used for Figure 3. To simplify the results, the base construct (K336CLM) is slightly different from that of Figure 2 (K349CLM in which a short coiled-coil part (337–349) exists). (B) As short dsDNA can be treated as a rod, the area accessible on the MT by the detached head is restricted to a doughnut-shaped area (left). The width of the doughnut-shaped area is constant for various DNA lengths (right). Taken together, we can control the area accessible by the detached head. For example, with short DNA the detached head can reach the next binding site (closed arrow head); with long DNA it can reach a binding site a two-step distance away (open arrow head). Note: DNA is rigid in the longitudinal direction, but the carbon linkers between DNA and the head ensure flexibility in the rotational direction. Thus the collision probability of the binding surface of the head is not restricted to the rotational direction (see Supplementary Results). (C) A kymograph of the Cy5 channel showed that anti-parallel DNA-kinesin also walked processively for various DNA lengths. Scale bar for vertical axis=4 s, horizontal axis=4 μm. (right bottom) Enlarged kymograph of 25 bp constructs. Motile molecules are encapsulated by yellow dashes. (D) Motile probability shows dependence on DNA length. Unexpectedly, motile probability at the two-step distance (arrow) is low. Note: the peak was expected at a DNA length of 8 nm (16 nm−length of native neck linker length (8 nm)).

Figure 4

Motile properties of anti-parallel type DNA-kinesin. Data of the construct connected at position 337 was analyzed more precisely. (A) Velocity (left) and run length (right) profiles show dependence on DNA length. (B) To compare, we plotted the relative values against DNA length. Data was normalized using the data from the 6 bp constructs (2.4 nm). Run length (blue squares), residence time (open triangles; original data not shown) and motile probability (open diamonds; data from Figure 3 of main text) decreased faster than velocity (red circles). To obtain the run length and residence time, data were fitted by a nonlinear least squares fitting of the cumulative probability distribution [C1^*(1−exp (−t/C2))−C3 from t=0 to infinity] where C1 is a normalized parameter and C2 is the run length or the residence time. C3 was used to exclude the effect of the counting loss.

Figure 5

Evaluating the contribution of the power stroke and communication through the neck linker. (A) Dual function of the neck linker has been proposed to facilitate processive runs. (B) To evaluate the contribution of the two functions independently, four conditions were compared. ^*Connection at the root of the neck linker (position 324; also see Supplementary Results). (C) The neck linker undergoes a nucleotide-dependent conformation change and so attaches to (docking) or detaches from (undocking) the head. (D) The neck linker is thought to act as a lever arm. By connecting the head to the midpoint of the neck linker, we could evaluate the effect of the effective lever arm length (bias length). For example, a small bias length is expected for connections at 324, whereas a large bias length is expected for connection at 337. Closed circle (324) and closed square (337) show the positions for the undocked state; open circle (324) and open square (337) show the positions for the docked state. A docked state crystal structure is shown (PDB entry 1MKJ).

Figure 6

The C-terminal neck linker sequence has dual function. (A) To resolve the coordination mechanism especially with respect to uncover the contribution of the neck linker on processive movement, we compared several constructs with different connect positions. (B) Connect positions. The amino acid number is equal to that of human kinesin. N- and C-termini are colored blue and red, respectively. Positions in which fluorescent dye labeling had no effect on movement were labeled with DNA. Note: all constructs had a full-length neck linker. (C) Position dependence of movement. Note: the distance between the connect positions is different for different positions, thus the peak position changes. (D) Observed max motile probability for each connect position. Data of position 337 is from Figure 3. Note: displacement analysis showed that N-terminal connect constructs (position 2 and 7) moved unidirectionally, but Mid connect constructs (23–215) moved bi-directionally. Taken together with the mean square displacement (MSD) analysis, we concluded that N-terminal constructs move processively and Mid constructs move by diffusion (see Supplementary Figure S6). (E) Neck linker docking and C-terminal connection are critical for efficient movement. Bias length was calculated from the crystal structure (1MKJ) assuming position 324 as the starting point. ‘comm' in the figure means Cterm communication. Note: from the crystal structure study, the N-terminal of kinesin is known to attach to the neck linker in a docked state. So for the N-terminal (blue symbols), data with (•) or without (x) attachment to the neck linker is plotted. Data from C-terminal and Mid positions are plotted with red and green circles, respectively. (F) Dual functions of the neck linker are crucial for processive movement.

Figure 7

Internal strain affects run length more than velocity. (A) Parameters for calculating internal strain. D, distance between two connect positions, L_DNA, length of DNA (end-to-end distance). See Materials and methods section for details. (B) Velocity depends on the estimated internal strain. Data for connect positions 328, 333 and 337 were shown (carbon spacer is EMCS; see 7C). Note: the estimated value of the internal strain depends on the model, but we assume that the qualitative trend is independent of the model. (C) To compare the different dependencies of run length and velocity on internal strain, we changed the carbon chain spacers, which connect DNA and kinesin head (see text for details). We used three spacers: AMAS, EMCS and KMUS. (D) Data of the three carbon spacers are shown (connect position is 328). Motile probability of different constructs showed different peak positions. This result, namely that a short linker needs longer DNA and a long linker needs shorter DNA, is reasonable. (E) The internal strain affects run length more than velocity. Note: to simplify the interpretation, only DNA longer than the optimal motile length were plotted (data of some short DNA conditions (6 and 7 bp for AMAS, 6 bp for EMCS) were not plotted).

Figure 8

Applying strain on the back domain of the head is the origin of the head–head coordination. (A) To resolve the origin of the coordination, we compared several constructs with different connect positions. To do this, two monomers at different connect positions were dimerized: one head (head A) is connected at the location of interest while the other head (head B) is connected at the end of neck linker to achieve forward bias. (B) Connect positions. (C) Position dependence of the movement. Note: From displacement and MSD analysis, we concluded that 23 and 324 constructs move processively (see Supplementary Figure S7). (D) Dual function of the neck linker is crucial for processive movement, as internal strain applied to the rear of the leading head prevents simultaneous detachment of both heads from the MT.