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

2009; Springer Nature; Volume: 29; Issue: 1 Linguagem: Inglês

10.1038/emboj.2009.319

1460-2075

Yuya Miyazono, Masahito Hayashi, Peter Karagiannis, Yoshie Harada, Hisashi Tadakuma,

Microtubule and mitosis dynamics

Article5 November 2009Open Access Strain through the neck linker ensures processive runs: a DNA-kinesin hybrid nanomachine study Yuya Miyazono Yuya Miyazono Department of Applied Physics, The University of Tokyo, Tokyo, Japan Search for more papers by this author Masahito Hayashi Masahito Hayashi The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Peter Karagiannis Peter Karagiannis Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Yoshie Harada Yoshie Harada The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan Search for more papers by this author Hisashi Tadakuma Corresponding Author Hisashi Tadakuma Department of Applied Physics, The University of Tokyo, Tokyo, Japan Graduate School of Frontier Science, The University of Tokyo, Chiba, Japan Search for more papers by this author Yuya Miyazono Yuya Miyazono Department of Applied Physics, The University of Tokyo, Tokyo, Japan Search for more papers by this author Masahito Hayashi Masahito Hayashi The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Peter Karagiannis Peter Karagiannis Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Search for more papers by this author Yoshie Harada Yoshie Harada The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan Search for more papers by this author Hisashi Tadakuma Corresponding Author Hisashi Tadakuma Department of Applied Physics, The University of Tokyo, Tokyo, Japan Graduate School of Frontier Science, The University of Tokyo, Chiba, Japan Search for more papers by this author Author Information Yuya Miyazono1, Masahito Hayashi2, Peter Karagiannis3, Yoshie Harada2,4 and Hisashi Tadakuma 1,5 1Department of Applied Physics, The University of Tokyo, Tokyo, Japan 2The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 3Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan 4Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan 5Graduate School of Frontier Science, The University of Tokyo, Chiba, Japan *Corresponding author. Graduate School of Frontier Science, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan. Tel.: +81 4 7136 3648; Fax: +81 4 7136 3648; E-mail: [email protected] The EMBO Journal (2010)29:93-106https://doi.org/10.1038/emboj.2009.319 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Conventional kinesin (kinesin-1; hereafter referred to as kinesin) is a motor protein, which drives cellular transport using the energy derived from ATP (Vale and Milligan, 2000; Hirokawa and Takemura, 2005). Kinesin's head (catalytic domain), which binds to microtubules (MTs) and hydrolyzes ATP, is located at the N-terminus of the polypeptide, whereas two identical heads are dimerized through the C-terminal coiled-coil. Kinesin walks processively along the MT over long distances (more than 1 μm) with an 8-nm step that matches the repeat distance of the MT lattice, and can generate a force of up to 7 pN (Svoboda et al, 1993; Vale et al, 1996). When walking, kinesin alternately repeats one-head and two-head bound states to move in a 'hand-over-hand' manner (Asbury et al, 2003; Kaseda et al, 2003; Yildiz et al, 2004). To walk efficiently over long distances, (1) the trailing head, but not the leading head, must detach from the MT at the end of the two-head bound state and (2) this detached head must bind forward, at the end of the one-head bound state. For the second condition, a bias mechanism regulated by the 'neck linker', a 13-amino-acid chain (residues 324–336) positioned at the C-terminal part of the head, has been proposed ('Power stroke model') (Rice et al, 1999). For detachment of the trailing head, a regulation mechanism that coordinates both heads has been proposed (for reviews, see Block, 2007; Hackney, 2007). The power stroke model was originally proposed for the motor protein myosin (Huxley, 1969; Huxley and Simmons, 1971). In myosin, a chemical state change in the bound nucleotide generates a small mechanical conformational change in the head. This small conformational change is amplified by a rigid lever arm. The power stroke of the lever arm shifts the detached head to bias its reattachment to the next forward actin filament-binding site (Spudich, 1994, 2001; Dunn and Spudich, 2007; Shiroguchi and Kinosita, 2007). In the myosin power stroke, the length of the lever arm, which contributes to the forward displacement of the detached head, is important for directional movement (Purcell et al, 2002; Sakamoto et al, 2003, 2005). Kinesin lacks the rigid lever arm, but upon ATP binding to the head, a conformational change in the neck linker occurs during which the neck linker binds to the head (termed 'docking'). This docking is believed to be analogous to the myosin lever arm enabling a similar walking mechanism (Rice et al, 1999; Case et al, 2000; Tomishige and Vale, 2000; Tomishige et al, 2006). By docking the neck linker to the leading head, the C-terminus of the leading head is repositioned toward the MT plus end, thus shifting the position of the detached head forward. Although the contribution of the neck linker docking is well established, there are still some arguments regarding whether this docking powers kinesin movement (Nishiyama et al, 2001, 2002; Schief and Howard, 2001; Carter and Cross, 2005; Block, 2007). On the other hand, coordination of the heads ensures long-distance travel. To walk more than a hundred steps without dissociation, native kinesins coordinate the activities of their two heads such that one head always remains attached to the MT, while the trailing head, but not leading head, is always detaches from the MT. It is thought that there is a checkpoint (termed 'gating'), in which the walking cycle is stalled until a specific nucleotide binding or conformational change occurs. Several types of gating mechanisms have been proposed, although two models are most popular. One is the 'mechanical gate model' in which the power stroke of the leading head accelerates the detachment of the trailing head (Hancock and Howard, 1999), meaning the gating contributes to the velocity of the molecules (see Supplementary Discussion for details). The other is the 'chemical gate model' according to which ATP binding of the leading head is inhibited until detachment of the trailing head occurs, meaning only the trailing head can become weak binding and subsequently detach from the MT (Rosenfeld et al, 2003; Klumpp et al, 2004). Thus simultaneous detachment from the MT by both heads is prevented by gate mechanism such that gating contributes to run length, which is a measure of processsivity. In the two-head bound state for both models, the neck linker that connects the two heads is more or less fully extended. Thus the inter-head tension (or 'internal strain') is believed to be the origin of head–head communication. To distinguish between these two models, two groups attempted to reduce the internal strain by extending the length of the neck linker (Hackney et al, 2003; Yildiz et al, 2008). Hackney et al (2003) inserted additional peptide residues (1–12 residues per head) between the neck linker and the coiled-coil part finding that kcat and the multi-motor sliding velocity of axonemes remain constant. However, the kinetic processivity (defined as the number of ATPs hydrolyzed per productive microtubule encounter) in these mutants is significantly less than wild type. Furthermore, another biochemical study (Rosenfeld et al, 2003) showed that the ATPases of monomers, that have no internal strain, and dimers are identical, meaning ATPase acceleration due to dimerization is negligible. These results support the chemical gate model. However, recent single molecule experiments showed conflicting results (Yildiz et al, 2008). At the single molecule level, Yildiz et al (2008) observed constructs by inserting 2–26 polyproline or seven repeats of glycine–serine residues (14GS) into each head. These extended kinesins remained processive and their run length was almost unchanged. However, the velocity significantly decreased. Interestingly, the speed recovered to near normal levels when an external tension was applied to the motor by an optical trap along the direction of movement. As this tension was applied more to the trailing head than leading head, these results were interpreted to mean that trailing head detachment was promoted by external tension, suggesting that internal force generated by the leading head's docking promotes trailing head detachment during the normal walking cycle. Thus, single molecule experiments support the mechanical gate model. To resolve this discrepancy, a novel approach is needed. Another issue is the structural basis for the coordination. Recently, Yildiz et al (2008) showed that external tension can induce directional stepping in normally immobile kinesin constructs that lack both mechanical element (neck linker) and fuel (ATP). They proposed a hypothesis according to which the head itself can sense and respond to strain to ensure unidirectional movement. Resolving the sensing domain (or element) should provide important information regarding the coordination mechanism. However, using a classical method to construct neck linker mutants (point mutations, extensions and replacements) results in strain always being applied through the neck linker. Thus, it is difficult to conclude whether sensing tension is done by the whole head or by a specific domain. To clarify the mechanism unequivocally, one needs to explore precisely the effect of changing the distance between the two heads and applying strain to many locations on the head. However, a construct solely based on proteins cannot fulfill all these required conditions. Therefore, we constructed a DNA-kinesin hybrid nanomachine (hereafter 'DNA-kinesin') that connects the two monomers with DNA. Advantages using DNA are that short dsDNA can act as a rigid rod (Bustamante et al, 1994; Wang et al, 1997; Mathew-Fenn et al, 2008; see also Supplementary Results) and the DNA length can be changed incrementally 0.34 nm by changing one base, meaning one can control the distance between the two heads with sub-nanometer accuracy. In addition, by introducing a Cys residue, any surface position of the head can be labeled with DNA. Thus, both the connect position and the distance between the two heads can be fully controlled, so one can evaluate the head–head coordination mechanism precisely. Using this novel assay, we explored the origin of processive movement in kinesin. Results Construction and confirmation of DNA-kinesin For DNA-kinesin construction, a Cys residue was introduced at a specific position in the Cys light mutant (CLM), where fluorescent dye labeling has no effect on activity (Rice et al, 1999; Tomishige et al, 2006). Then fluorescently labeled DNA–maleimide was covalently attached. DNA-kinesin dimers were obtained by mixing two hetero DNA-kinesin monomers, in which one monomer had a sense sequence and the other had an antisense sequence (Figure 1A). Attachment of the kinesin head at the 5′ or 3′ end resulted in a parallel or anti-parallel type dimer. The parallel type mimics the coiled-coil part of kinesin, whereas the anti-parallel type resembles the layout of the neck linker. Biochemical assays confirmed the dimerization of the DNA-kinesin at the bulk level (Figure 1B and C show the data of anti-parallel type DNA-kinesin connected at the C-terminal end of the neck linker (position 337; see Figure 3A). Table I lists the constructs used). 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). Download figure Download PowerPoint Table 1. List of the constructs used in the Figures 1, 2, 3, 4, 5, 6, 7 Construct Position Feature (bias length) Figure 1 K336CLM 337 337(C-terminal) End of C-terminus (full) Figure 2 K349CLM 342 342(C-terminal) End of C-terminus (full) Figure 3 K336CLM 337 337(C-terminal) End of C-terminus (full) Figure 4 K336CLM 337 337(C-terminal) End of C-terminus (full) Figure 6 K336CLM 2 2(N-terminal) Tip of N-terminus (a,b, a,b) K336CLM 7 7(N-terminal) On the side of head a,b, a,b) K336CLM 23 23(mid) Back part of head (none) K336CLM 43 43(mid) Back part of head (none) K336CLM 101 101(mid) Dorsal part of head (none) K336CLM 215 215(mid) Front part of head (none) K336CLM 324 324(C-terminal) Root of C-terminus (none) K336CLM 328 328(C-terminal) Midpoint of C-terminus (partial) K336CLM 333 333(C-terminal) End of C-terminus (almost full) Figure 7 K336CLM 328 328(C-terminal) Midpoint of C-terminus (partial) K336CLM 333 333(C-terminal) End of C-terminus (almost full) K336CLM 337 337(C-terminal) End of C-terminus (full) Names of the constructs, points of DNA attachment and construct feature are listed. 'Bias length' is an indicator of effective lever arm length (see Figure 5D). a Partial or none. b From the crystal structure study (PDB entry 1MKJ), the N-terminal of kinesin is known to attach to the neck linker in a docked state. Therefore, the bias length might be partial or none (the latter corresponds to the state without attachment to the neck linker). In Figures 1, 2, 3, 4, 5, 6 and 7B, EMCS, which has six carbon chains, was used as the connection linker between DNA and kinesin. To further explore the effects of the DNA-kinesin connection linker, other bi-functional linkers (AMAS: 2 carbon chains; KMUS: 11 carbon chains) were used in Figure 7D and E. For Figure 8, K336CLM 337 was heterodimerized with the constructs used in Figure 6 (K336CLM 23, 43, 101, 215, 324). To assess the motile activity of DNA-kinesin at the single molecule level, we first observed the parallel type that replaced the coiled-coil of native kinesin with DNA at connect position 342, the C-terminal end of the neck linker (Figure 2B inset). With the parallel type, two fluorescent dyes (TAMRA and Cy5) were attached on the same side of the DNA at a short distance from each other. Thus, a high fluorescent resonance energy transfer (FRET) signal was expected (Figure 2A). On exciting these DNA-kinesin dimers using a green laser (514 nm), fluorescent spots in the TAMRA channel were rare, whereas motile fluorescent spots were only observed in the Cy5 channel (Figure 2B). The fluorescence intensity of the motile spots was the same as that of a single fluorophore. In addition, anti-correlative, simultaneous recovery of TAMRA fluorescence with photo bleaching of the Cy5 fluorophore was observed (Supplementary Figure S1B). From these results, we concluded that the motile fluorescent spots were those of single molecule DNA-kinesin. The velocity dependence on ATP concentration obeyed Michaelis–Menten kinetics, showing that the motility is an ATP-dependent process (Figure 2C). However, Vmax was 235 nm/s, half the speed of wild-type kinesin (K490CLM 416-Qdot655; 525 nm/s), and the run length (130 nm; Figure 2D) was 1/10 that of wild type (1.3 μm). A biochemical study (Hackney et al, 2003) and a recent single molecule study (Yildiz et al, 2008) have showed that the insertion of polyglycine or polyproline residues into the 'neck linker–coiled-coil interface', which changes the distance between the two heads, lowers the velocity and decreases the run length. This may also occur in DNA-kinesin, as parallel type constructs have a carbon chain spacer between the DNA and neck linker, which acts as a soft elastic linker (see Figure 7C for details). Another possible explanation for these results is the effect of charged residues. As the charged residues of a coiled-coil (e.g. Lys) greatly contribute to run length (Thorn et al, 2000), DNA-kinesin's short run length might be the result of the construct lacking the coiled-coil region. 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 Vmax (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. Download figure Download PowerPoint Effect of distance between heads To resolve the mechanism that coordinates the two heads in kinesin walking, we measured the motile activity of anti-parallel type constructs of varying DNA lengths (6–40 bp dsDNA) at the single molecule level. As the inter-head connection linker lengthens, the tense neck linker that is more or less fully extended in native kinesin is expected to relax. Similar approaches have been used previously with contradictory results (Hackney et al, 2003; Yildiz et al, 2008). These reports, however, used protein-only constructs that had soft peptide residues (polyglycine or glycine–serine repeats; persistence length lp=0.8 nm; Sahoo et al, 2006), or semi-rigid polyproline (lp=4.4 nm; Schuler et al, 2005) insertions. These polypeptides, though, cannot be treated as rigid rod, as they behave as elastic springs (see Supplementary Results for details). Furthermore, even for semi-rigid polyproline, many free joints in the constructs exist, including those between the neck linker and inserted polypeptides and those between the polypeptides and coiled-coil. Thus, by increasing the number of inserted peptides, both the mean distance between the heads and the area on the MT the heads can access change. This might cause an increase in the number of futile steps (e.g. side steps or back steps). For less equivocal data, we observed anti-parallel type DNA-kinesin that resembles the layout of an extended neck linker (Figure 3A), but with a rod-like backbone made of DNA resulting in unique characteristics. For example, in DNA-kinesin, the mean distance between the two heads can be changed in 0.34-nm increments by changing the number of nucleotides in the rod-like DNA. As short dsDNA is a rigid rod (lp=50 nm, Bustamante et al, 1994), the area accessible on the MT by the detached head, which corresponds to the width of the doughnut in Figure 3B, is constant (see also Supplementary Figure S8C). Thus we could measure the effect of the distance between the heads more accurately using DNA-kinesin than that of a protein-only construct (see Supplementary Figures S8–S10 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)). Download figure Download PowerPoint We measured the effect of the distance between the heads with a construct connected at position 337. Here, docking of the neck linker is not disturbed. Thus, the detached head should swing forward the length of the neck linker. Therefore, if the distance between the heads is the same as that of wild type, processive movement of DNA-kinesin should be observed. However, when the DNA length is changed, the motile probability, which is defined as 'the number of motile molecules/number of molecules attached to MT' and is a rough indicator for run length (see Figure 4B and Supplementary Figure S4B), should decrease as the DNA-kinesin's distance between the heads becomes longer than that of wild type. This is because too long a length lowers accessibility to the next MT binding site that is only 8 nm away. However, further extending the distance between the heads to 16 nm might actually increase accessibility, as the head can now reach the second consecutive binding site (16 nm away). Therefore, a local maximum for the motile probability was expected at a DNA length of 8 nm (=16 nm−original length of the neck linker (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. Download figure Download PowerPoint After preparation of the 337 construct, we measured the end-to-end distance of DNA. At both DNA ends, one of two fluorophores (Cy3 or Cy5) was attached. As the two fluorophores were close, the FRET signal was expected to depend on the distance between the two. The observed FRET efficiencies of many DNA lengths were similar to those values predicted from the duplex structure (Supplementary Figures S2 and S3), suggesting that we could control the end-to-end DNA distance, as expected. On measuring the motility of DNA-kinesin at variable DNA lengths (Figure 3C), the motile probability was observed to become lower as the DNA became longer, with motile molecules being negligible at a DNA length of 6 nm (=18 bp). Further extension allowed some molecules to take steps equivalent to a two-step distance, although the probability was unexpectedly low (5%, Figure 3D). At present we do not know the reason for this low probability. Recently, Yildiz et al (2008) reported that extending the neck linker length by inserting polyproline allows occasional side steps because of the linker's long reach. These steps are futile for processive movement and decrease the coordination between the heads. This may also occur upon DNA lengthening (see Supplementary Discussion for details). To evaluate more precisely, we measured the velocity and the run length of each DNA length (Figure 4). Our DNA-kinesin results show that extending the distance between the heads by changing the length of DNA, while keeping the connect position constant (position 337), caused the run length to shorten, but the velocity to remain constant. These results are similar to those of a biochemical study (Hackney et al, 2003), but are in contrast to those from a recent single molecule study (Yildiz et al, 2008). To explain these contradicting results and better resolve the coordination mechanism, we took advantage of the unique characteristics of DNA-kinesin. Exploring the coordination mechanism As the neck linker connects the two heads, communication between the heads should be transmitted through the neck linker. However, it is believed that the function of the neck linker (C-terminal linker) is not limited to inter-head communication (hereafter such communication is termed 'Cterm communication'), but also involved in biasing the detached head forward by docking (Figure 5A). Thus, to evaluate the contribution of the neck linker on the head–head coordination mechanism, we needed to evaluate the power stroke and Cterm communication independently. To do this, we evaluated the effects of both by changing both the connect position and the distance between the heads using constructs with full-length neck linkers. We compared the following four conditions (Figure 5B): (1) both the power stroke and Cterm communication are active, (2) only the power stroke is active, (3) only Cterm communication is active and (4) both mechanisms are inactive. Constructs in which two monomers are connected at the C-terminus (neck linker) correspond to condition 1. A crystal structure study (PDB: 1MKJ) and molecular dynamics simulation (Hwang et al, 2008) showed that in the docked state, the N-terminus attaches to the neck linker through a β-sheet structure (Figure 5C). However, in the undocked state, a crystal structure (PDB: 1BG2) showed no sign of N-terminal residues, suggesting the N-terminus was detached. Thus, constructs connected to N-terminal residues were expected to correspond to condition 2, in which the power stroke arose from docking of the neck linker without Cterm communication. Construct connected at the root of the neck linker (position 324) corresponded to condition 3, where Cterm communication arose from the neck linker, without any forward bias of the detached head despite the power stroke (Figure 5D). Having the connection point at the midway point of the neck linker sequence (positions 333 and 328; see Figure 6A and B) allows the detached head to undergo free diffusion with the connect position acting as a pivot point. Thus, on neck linker docking, amino acids from the root of the neck linker to the connect position function as an effective lever arm, and the detached head is biased forward a distance ranging